REVIEW
published: 14 January 2019
doi: 10.3389/fgene.2018.00722
Frontiers in Genetics | www.frontiersin.org 1 January 2019 | Volume 9 | Article 722
Edited by:
Akio Kanai,
Keio University, Japan
Reviewed by:
Yutaka Hirose,
University of Toyama, Japan
Daisuke Kaida,
University of Toyama, Japan
Florian Grebien,
Ludwig Boltzmann Institute for Cancer
Research (LBI-CR), Austria
*Correspondence:
Akihiko Yokoyama
ayokoyam@ncc-tmc.jp
Specialty section:
This article was submitted to
RNA,
a section of the journal
Frontiers in Genetics
Received: 29 October 2018
Accepted: 21 December 2018
Published: 14 January 2019
Citation:
Yokoyama A (2019) RNA Polymerase
II-Dependent Transcription Initiated by
Selectivity Factor 1: A Central
Mechanism Used by MLL Fusion
Proteins in Leukemic Transformation.
Front. Genet. 9:722.
doi: 10.3389/fgene.2018.00722
RNA Polymerase II-Dependent
Transcription Initiated by Selectivity
Factor 1: A Central Mechanism Used
by MLL Fusion Proteins in Leukemic
Transformation
Akihiko Yokoyama*
Tsuruoka Meatabolomics Laboratory, National Cancer Center, Yamagata, Japan
Cancer cells transcribe RNAs in a characteristic manner in order to maintain their
oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA
polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively).
The transcriptional machinery that initiates each transcription reaction has been purified
and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1
pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent
transcription remains unclear. Our group previously found that SL1 specifically associates
with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family
proteins and the P-TEFb elongation factor. Similar complexes have been independently
characterized by several different laboratories and are often referred to as super
elongation complex. The involvement of AEP in RNAP2-dependent transcription
indicates that SL1 must play an important role in RNAP2-dependent transcription. To
date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family
genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant
MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP
components. The MLL portion functions as a targeting module, which specifically binds
chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs.
This type of chromatin is enriched at the promoters of transcriptionally active genes
which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich
gene promoters. The fusion partner portion, which recruits other AEP components and
SL1, is responsible for activation of RNAP2-dependent transcription. Consequently,
MLL fusion proteins constitutively activate the transcription of previously-transcribed
MLL target genes. Structure/function analysis has shown that the ability of MLL
fusion proteins to transform hematopoietic progenitors depends on the recruitment
of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears
to be the central mechanism of MLL fusion-mediated transcriptional activation.
However, the molecular mechanism by which SL1 activates RNAP2-dependent
Yokoyama SL1 Mediates RNAP2-Dependent Transcription
transcription remains largely unclear. This review aims to cover recent discoveries of the
mechanism of transcriptional activation by MLL fusion proteins and to introduce novel
roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery
may be involved in RNAP2-dependent gene regulation.
Keywords: RNA polymerase, SL1, transcription, leukemia, MLL, AEP, DOT1L
EUKARYOTES HAVE THREE MAJOR RNA
POLYMERASES
In prokaryotes, one RNA polymerase transcribes all genes.
Eukaryotic cells contain multiple RNA polymerases, which
transcribe different classes of genes (Roeder and Rutter,
1969; Thomas and Chiang, 2006; White, 2008; Vannini
and Cramer, 2012; Turowski and Tollervey, 2016; Khatter
et al., 2017; Zhang et al., 2017). Most genes are transcribed
by three major RNA polymerases, RNA Polymerase I,
II, and III (RNAP1, RNAP2, and RNAP3, respectively).
RNAP1 transcribes pre-rRNA, which is later processed
into three large rRNA species, 28S, 18S, and 5.8S. RNAP2
transcribes protein-coding genes to yield mRNAs. RNAP3
transcribes 5S rRNA and tRNAs. Small nuclear RNAs and small
cytoplasmic RNAs are transcribed by either RNAP2 or RNAP3
(Figure 1).
RNA POLYMERASE I
RNAP1 synthesizes the 47S pre-rRNA, which is processed into
mature 28S, 18S, and 5.8S rRNAs (Goodfellow and Zomerdijk,
2013; Khatter et al., 2017; Zhang et al., 2017). The Human
genome contains ∼400 rDNA repeats, about 50% of which are
transcribed, accounting for up to 60% of the entire transcriptional
activity (Birch and Zomerdijk, 2008; Schlesinger et al., 2009).
Pre-initiation complex (PIC) formation of RNAP1 at the rDNA
promoter is triggered upon association of selectivity factor 1
(SL1) with the core promoter, in the presence of UBF (Learned
et al., 1985; Eberhard et al., 1993; Comai et al., 1994; Zomerdijk
et al., 1994; Heix et al., 1997; Gorski et al., 2007) (Figure 1A).
SL1 comprises TBP, TAF1A (also known as TAFI48), TAF1B
FIGURE 1 | Transcriptional initiation complexes of the three eukaryotic RNA polymerases. The pre-initiation complexes (PIC) of RNA Polymerase I (A), II (B), and III (C)
are depicted. (A) In the presence of UBF, SL1 recognizes the rDNA promoter and recruits RNAP1. TBP binding induces a bend in the DNA, which leads to the
formation of PIC. (B) TBP binding and subsequent recruitment of RNAP2 and other auxiliary factors completes the formation of PIC. Mediator is thought to be
involved in PIC stabilization and in the initiation of transcription. (C) At tRNA promoters, TFIIIC located in the intragenic region recruits TFIIIB, which contains BDP1 and
BRF1, to further recruit RNAP3.
(also known as TAFI63), TAF1C (also known as TAFI110),
and TAF1D (also known as TAFI41) and recruits the RNAP1
complex to induce PIC formation. It is thought that all three
polymerases employ similar mechanisms to start transcription
(Naidu et al., 2011; Vannini and Cramer, 2012; Khatter et al.,
2017), in which the universal role of TBP is to bend the template
DNA, while TAF1B, TFIIB, and BRF1 proteins respectively
recruit corresponding polymerases to the promoter.
RNA POLYMERASE II
RNAP2 has been most rigorously studied and shown to
collaborate with various associated factors in a step-wise manner
to transcribe mRNAs (Roeder, 1996; He et al., 2013). Initiation
of in vitro basal transcription on a model promoter starts with
loading of the TATA binding protein (TBP) to the TATA box
(Basehoar et al., 2004), which is positioned approximately 25
nucleotides upstream of the transcription start site (Roeder, 1996;
He et al., 2013). TBP binding induces a bend in the double
helix (Kim J. L. et al., 1993; Kim Y. et al., 1993) and recruits
TFIIB to stabilize the DNA/protein complex (Figure 1B). TFIIB
then recruits RNAP2 and TFIIF to form a PIC (Roeder, 1996;
He et al., 2013). The initiation of transcription requires the
recruitment of TFIIE and TFIIH. TFIIH unwinds DNA at the
initiation site and phosphorylates the Ser 5 residue of the RNAP2
C-terminal domain heptapeptide repeat to release the polymerase
from the PIC. In vivo RNAP2-dependent transcription is much
more complicated. TBP binds to various TBP-associated factors
(TAFs) to form a large complex called TFIID, which facilitates
promoter recognition, especially at promoters lacking an obvious
TATA box (Dynlacht et al., 1991; Pugh and Tjian, 1991). Gene
promoters with a TATA box tend to be bound by the SAGA
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
complex which includes TBP, SUPT3H, and GCN5 (Basehoar
et al., 2004; Rodríguez-Navarro, 2009). Therefore, it was thought
that TATA-containing genes were mainly regulated by the SAGA
complex, while TATA-less genes were independently regulated by
TFIID (Pugh and Tjian, 1991; Basehoar et al., 2004). However,
recent studies in yeast indicate that most genes utilize both TFIID
and SAGA, and that the relative contribution of each complex
likely depends on the individual context (Baptista et al., 2017;
Warfield et al., 2017). The Mediator co-activator complex is also
involved in transcription initiation for the expression of nearly all
genes (Malik and Roeder, 2010; Warfield et al., 2017). Mediator
disruption caused more severe defects than did the disruption
of TFIID subunits, suggesting that there may be a low level of
TFIID-independent transcription at many genes that is derived
from PICs assembled with TBP and lacking TAFs. Nearly all
RNAP2-regulated genes, with or without a TATA box in the
promoter, are thought to use TBP for transcriptional activation.
RNA POLYMERASE III
RNAP3 transcribes 5S rRNA, tRNAs, and various small noncoding
RNAs (White, 2008; Vannini and Cramer, 2012; Turowski
and Tollervey, 2016; Khatter et al., 2017). The clearest feature of
RNAP3 transcripts is that they are all untranslated and less than
300 base pairs in length. tRNA gene transcription requires TFIIIB
and TFIIIC (Figure 1C). TFIIIC binds to intragenic elements
and positions TFIIIB onto the tRNA promoter. TFIIIB, which
contains TBP and BRF1, then induces PIC formation to start
RNAP3 transcription.
RNA POLYMERASE I/SL1-DEPENDENT
RIBOSOMAL RNA TRANSCRIPTION IN
CANCER
In cancer cells, rRNA transcription is upregulated. This increases
the cell’s ability to produce proteins to meet the metabolic
demands of quickly proliferating cancer cells (White, 2008).
High level pre-rRNA expression is observed in cancer cells and
is correlated with tumor stage (Williamson et al., 2006). MYC
is highly expressed in most cancer cells and upregulates cell
cycle-related genes and metabolism-related genes to promote cell
division and anabolism (Dang, 2012). MYC also activates rRNA
transcription directly (Arabi et al., 2005; Grandori et al., 2005;
Shiue et al., 2009) and indirectly by activating UBF expression
(Poortinga et al., 2004). PTEN, which is often inactivated in
cancer, represses RNAP1-dependent transcription by disrupting
the SL1 complex (Zhang et al., 2005). Thus, loss of PTEN
facilitates cancer specific metabolism in part by enhancing SL1mediated
rRNA transcription.
AF4 FAMILY/ENL FAMILY/P-TEFB
COMPLEX
Unexpectedly, our group identified SL1 as a specific interactor
of AF4 (also known as AFF1), which is involved in RNAP2dependent
transcriptional activation (Okuda et al., 2015). This
result indicated that SL1 is involved in both RNAP1- and
RNAP2-dependent transcription. AF4 is a member of the AF4
protein family that is composed of AF4, AF5Q31 (also known
as AFF4), LAF4 (also known as AFF3), and FMR2 (also known
as AFF2). Previously, we purified a protein complex nucleated
by AF4 and identified AF5Q31, ENL (also known as MLLT1),
CDK9, and CyclinT1 (also known as CCNT1) as its components
(Yokoyama et al., 2010) (Figure 2A). CDK9 and CyclinT1
form a complex called P-TEFb, which activates transcription
elongation by phosphorylation of the RNAP2 complex paused
by negative elongation factors such as DSIF an NELF (Wada
et al., 1998a,b; Yamaguchi et al., 1999; Peterlin and Price,
2006). We named this complex the AF4 family/ENL family/PTEFb
complex (AEP) (Yokoyama et al., 2010). ELL, which
retains transcription elongation activity (Shilatifard et al., 1996),
was also shown to associate with the AF4 family protein and
other AEP components, and this complex is often referred
to as the super elongation complex (Lin et al., 2010). Similar
complexes were independently identified and characterized in
several laboratories and have been shown to activate the RNAP2dependent
transcription elongation step for a subset of genes
including heat shock protein genes and the HIV viral genome
(He et al., 2010; Lin et al., 2010; Sobhian et al., 2010). Therefore,
it is thought that AEP activates transcription by activating
transcription elongation (Luo et al., 2012).
AF4 FAMILY PROTEINS ACTIVATE
TRANSCRIPTION VIA SL1
AF4 family proteins have been shown to activate RNAP2dependent
transcription (Prasad et al., 1995; Ma and Staudt,
1996; Morrissey et al., 1997; Hillman and Gecz, 2001). GAL4dependent
transactivation assays use reporter gene expression to
indicate activation of transcription. Reporter gene transcription,
such as that of the firefly luciferase gene, is driven by a minimum
promoter tethered to multiple GAL4 responsive elements and is
measured in the presence of the GAL4 DNA binding domain
fused with the domain being tested. Using this approach,
the serine-rich pSER domain of AF4 protein family (Nilson
et al., 1997) was shown to activate transcription (Okuda et al.,
2015) (Figure 2B). In contrast, modules presumed to activate
transcription elongation by recruiting P-TEFb or ELL, did not
exhibit transactivation activity. These results indicate that, in
addition to recruiting elongation factors, AF4 family proteins
possess transcriptional activation functions. Assuming that the
pSER domain associates with transcriptional coactivators, our
group purified proteins associated with the GAL4-pSER fusion
protein and identified SL1 as a specific pSER domain binding
factor. Chromatin immunoprecipitation coupled with deep
sequencing (ChIP-seq) analysis in HEK293T cells showed that
TAF1C of SL1 co-localizes with AEP components in the vicinity
of transcription start sites of AEP target genes that are transcribed
by RNAP2 (Okuda et al., 2017). Knockdown of TAF1C resulted
in decreased expression of AEP target genes (defined as the genes
whose expression is reduced by ENL knockdown) (Okuda et al.,
2015). Deletion of the TATA box in the luciferase reporter cassette
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
FIGURE 2 | AEP-dependent transcriptional activation. (A) Composition of the AEP complex (AF4 family/ENL family/P-TEFb complex). AF4 and AF5q31 can form a
tero dimer and associate with ENL, CyclinT1, and CDK9. (B) Schematic representation of the AF4 and AF5Q31 protein structures. Protein-protein interaction is shown
by a dotted line. NHD, N-terminal homology domain; ALF, AF4/LAF4/FMR2 homology domain; pSER, poly-serine. A9ID, AF9 interaction domain; CHD, C-terminal
homology domain; (C) A hypothetical model of AEP-dependent transcriptional activation. First, AEP recruits SL1 and loads TBP onto the promoter through the SDE
and NKW motifs. TBP loading induces a bend. SL1 is then dismantled while TFIIB replaces TAF1B. AEP recruits Mediator through MED26 to facilitate transcription
initiation. P-TEFb activates transcription elongation of the RNAP2 complex paused by negative elongation factors such as DSIF and NELF. DSIF: DRB sensitivity
inducing factor. NELF: Negative elongation factor.
resulted in loss of pSER domain-mediated transactivation. Taken
together, these results suggest that the pSER domain first recruits
SL1 and then loads TBP to the TATA box to activate RNAP2dependent
transcription initiation.
MED26 POTENTIATES AF4-DEPENDENT
TRANSCRIPTION INITIATION BY SL1
RNAP2 PIC formation is facilitated by Mediator, a large protein
complex composed of ∼30 subunits (Malik and Roeder, 2010).
Mediator exists in a variety of subunit compositions, most of
which are conserved from yeast to metazoans. MED26, one of a
few metazoan-specific Mediator subunits, was shown to associate
with AEP in addition to associating with other Mediator complex
components (Takahashi et al., 2011). The pSER domain can
be divided into three subdomains, each of which contains one
evolutionarily conserved motif, DLXLS, SDE, and NKW (Okuda
et al., 2016) (Figure 2B). The DLXLS motif is a binding platform
for MED26 while the SDE motif is responsible for binding to SL1.
Although the function of the NKW motif remains unclear, it is
required for transactivation and is therefore postulated to play a
role in RNAP2-dependent transcription processes (Okuda et al.,
2015). The SDE and NKW motifs are necessary and sufficient to
activate transcription in GAL4-dependent transactivation assay.
The DLXLS motif is dispensable but can enhance SL1-mediated
transcription, presumably by recruiting Mediator (Okuda et al.,
2016). These results indicate that AEP activates transcription
initiation primarily via SL1, which can be further potentiated
by Mediator. This is contradictory to the current view that
AEP is specialized to transcription elongation. Hence, I propose
that AEP is a multi-functional transcriptional machinery that
can activate both initiation and elongation of transcription
(Figure 2C).
AEP-DEPENDENT TRANSACTIVATION IS
THE CENTRAL MECHANISM USED BY
MLL FUSION PROTEINS IN
LEUKEMOGENESIS
AEP components are frequent targets for chromosomal
translocation with the MLL gene (also known as KMT2A, HRX,
MLL1, HTRX, and ALL1) (Ziemin-van der Poel et al., 1991;
Djabali et al., 1992; Tkachuk et al., 1992; Nakamura et al., 2002;
Li and Ernst, 2014; Winters and Bernt, 2017; Yokoyama, 2017).
MLL is a transcriptional regulator that retains transcriptional
activation activity and histone methyltransferase (HMT) activity
and involved in transcriptional maintenance of Homeobox
(HOX) genes (Zeleznik-Le et al., 1994; Yu et al., 1995, 1998;
Ernst et al., 2001; Milne et al., 2002; Nakamura et al., 2002).
The resultant MLL-AEP fusion proteins cause aggressive acute
leukemia (Figure 3A) (Krivtsov and Armstrong, 2007; Li and
Ernst, 2014; Winters and Bernt, 2017). Leukemia involving
MLL gene rearrangements (MLL-r leukemia) is the cause of
5–10% of all acute leukemia cases (Meyer et al., 2018) and
is generally associated with poor prognosis (Rowley, 2008).
MLL-r leukemia cells express a subset of genes including
HOXA9 and MEIS1 whose expression is normally confined
to immature hematopoietic cells such as hematopoietic
stem cells (HSCs) (hereafter we refer to as HSC program genes)
(Armstrong et al., 2002; Yeoh et al., 2002; Krivtsov et al., 2006;
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
FIGURE 3 | General mechanism of leukemogenesis by MLL fusion proteins. (A) Schematic representation of the structures of wild type MLL and the MLL fusion
protein. The PWWP domain of LEDGF recognizes di-/tri-methylated histone H3 lysine 36. The CXXC domain of MLL binds to unmethylated CpGs. The PHD finger 3
binds to di-/tri-methylated Histone H3 lysine 4 (H3K4me2/3). The minimum targeting module necessary for target recognition (MTM) comprises the PWWP and CXXC
domains. HBM, HCF binding motif; PS, processing site; AD, activation domain; FYRN, FY-rich domain N-terminal; FYRC, FY-rich domain C-terminal. (B) Constitutive
activation of HSC program genes in leukemogenesis. Expression of HSC program genes progressively decreases during normal differentiation. However, MLL fusion
proteins constitutively activate HSC program genes to cause leukemia. (C) Ratio of fusion partners in MLL-r leukemia cases. Relative frequency of each fusion partner
is shown in a pie chart (adopted from the report from Meyer et al., 2018). AEP components such as AF4, AF9, and ENL account for two-thirds of MLL-r leukemia.
(D) A model of target recognition by MLL fusions proteins. First, the MLL fusion protein associates with MENIN, while LEDGF binds to nucleosomes containing
H3K36me2/3 marks. Next, the MLL fusion/ MENIN complex forms a stable complex with LEDGF on the promoter of HSC program genes. The MTM-ENL fusion
protein binds the same promoters as MLL fusion proteins.
Somervaille and Cleary, 2006) (Figure 3B). Sustained expression
of HOXA9 and MEIS1 in hematopoietic progenitors causes
leukemia in mouse models (Kroon et al., 1998), suggesting that
these two genes are strong drivers of leukemogenesis. MLL fusion
proteins directly bind the promoters of these HSC program genes
and constitutively activate their transcription (Ayton and Cleary,
2003; Somervaille and Cleary, 2006; Garcia-Cuellar et al., 2016;
Kerry et al., 2017; Okuda et al., 2017). Thus, the MLL fusion
protein is a constitutively-active transcriptional machinery that
transforms hematopoietic progenitors by aberrantly activating
HSC program genes (Krivtsov et al., 2006; Yokoyama, 2017).
Although MLL fuses with more than 100 different fusion partners
(Meyer et al., 2018), AEP components constitute two-thirds of
MLL-r leukemia cases (Figure 3C), indicating that merging the
functions of MLL and AEP is the most efficient way to generate
powerful leukemic oncogenes. Among the AEP components,
AF4 is the most frequent fusion partner for MLL, while AF5Q31
and LAF4 also fuse with MLL in rare cases of leukemia (Ma and
Staudt, 1996; Taki et al., 1999; Meyer et al., 2018). The second
most frequent fusion partner is AF9 (also known as MLLT3)
(Meyer et al., 2018), which is a homolog of ENL. ENL and AF9
constitute the ENL protein family and form a fusion with MLL
in one-third of MLL-r leukemia cases. ELL, which also binds to
the AF4 family protein, fuses with MLL in leukemia (DiMartino
et al., 2000; Luo et al., 2001; Lin et al., 2010). These results
strongly indicate that the transcriptional activation function of
AEP is the central mechanism utilized by MLL fusion proteins in
leukemogenesis.
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
TARGET CHROMATIN OF MLL AND MLL
FUSION PROTEINS
The role of the MLL portion of MLL fusion proteins is mainly
target recognition. Genome-wide ChIP-seq analysis showed that
MLL fusion proteins bound to the target chromatin of wildtype
MLL near transcription start sites (Guenther et al., 2005,
2008; Wang et al., 2011; Okuda et al., 2017). Most of the
target genes bound by MLL fusion proteins are included within
the list of MLL target genes (Milne et al., 2005; Wang et al.,
2011), and some genes are exclusively regulated by wild-type
MLL (Artinger and Ernst, 2013; Li et al., 2013). Wild type
MLL retains various chromatin reader modules including plant
homeodomain (PHD) fingers and a Bromodomain, which are
lost in MLL fusion proteins (Figure 3A). Thus, the mode of
target recognition is expected to be somewhat different between
wild type MLL and MLL fusion proteins. For example, PHD
finger 3 has been shown to bind to di-/tri-methylated histone
H3 lysine 4 (H3K4me2/3) (Wang et al., 2010), which plays a
significant role in target recognition by wild type MLL (Milne
et al., 2010). These observations indicate that the MLL portion
retained by MLL fusion proteins confers the ability to recognize a
subset of, but not all of, wild-type MLL target genes. Whether the
presence of wild type MLL is required for MLL fusion-dependent
leukemic transformation has been a topic of discussion. One
study showed that the remaining wild type allele is required
for leukemogenesis (Thiel et al., 2010). But recently, another
study showed that MLL is dispensable, while MLL2 (also known
as KMT2B), the closest homolog of MLL, plays a major role
in sustaining leukemogenesis (Chen et al., 2017), indicating
complex redundancy and independency within the target genes
of MLL fusion proteins, MLL and MLL2.
MECHANISM OF TARGET RECOGNITION
BY MLL FUSION PROTEINS
The structural requirements of MLL fusion-dependent leukemic
transformation can be evaluated using the ex vivo myeloid
progenitor transformation assay (Lavau et al., 1997). In this
assay, a retrovirus carrying an MLL fusion gene is transduced
into murine bone marrow-derived immature hematopoietic
progenitors and the cells are cultured ex vivo in semi-solid
media containing the required cytokines. Transduction of a
functional MLL fusion gene results in the cells expressing high
Hoxa9 levels and continuing to proliferate after rounds of
replating, while non-transduced cells stop proliferating during
early passages (Ayton and Cleary, 2003). Transformed cells
can be cultured for more than 5 months and are considered
“immortalized.” Immortalization is an important feature of
leukemic transformation and reflects the aberrant self-renewal
of cancer cells. Using this assay, domains within the MLL-ENL
fusion that are required for transformation were identified (Lavau
et al., 1997; Slany et al., 1998; Ayton et al., 2004). The Nterminal
region upstream of the AT hooks was shown to be
required for transformation (Figure 3A). This region contains
a motif responsible for the strong association with MENIN
(hMBM: the high affinity MENIN binding motif) (Yokoyama
et al., 2004, 2005). MLL-MENIN association triggers further
association with LEDGF through the LEGDF binding domain
(LBD) (Yokoyama and Cleary, 2008; Huang et al., 2012).
LEDGF contains the PWWP domain, which specifically binds
the nucleosomes with di-/tri-methylated histone H3 lysine 36
(H3K36me2/3) (Eidahl et al., 2013; Okuda et al., 2014; Zhu
et al., 2016) (Figure 3D). Mutant constructs of MLL-ENL lacking
the hMBM or the LBD failed to transform myeloid progenitors
because sequential association of MENIN and LEDGF is critical
for leukemic transformation. However, an artificial construct
tethering the PWWP domain to the MLL-ENL mutant lacking
the hMBM transformed myeloid progenitors, indicated that
MENIN’s primary role is to incorporate the PWWP domain
into the MLL-ENL complex and that other structures of
MENIN and LEDGF are dispensable (Yokoyama and Cleary,
2008). Further structure/function analysis demonstrated that
only three domains of the MLL-ENL complex are required for
leukemic transformation: the PWWP domain of LEDGF, the
CXXC domain of MLL, and the ENL portion (Okuda et al.,
2014) (Figures 3A,D). The CXXC domain specifically binds
to unmethylated CpGs (Birke et al., 2002; Allen et al., 2006;
Cierpicki et al., 2010). Because an artificial construct composed
of the PWWP and CXXC can target the promoters of HSC
program genes, combination of these two domains is defined as
the minimum targeting module (MTM) (Figure 3A).
DI-/TRI-METHYLATED HISTONE H3
LYSINE 36
Many epigenetic modifiers possess PWWP domains. For
example, BRPF1 has a PWWP domain, which also binds to
H3K36me2 and H3K36me3 and can functionally substitute
for that of LEDGF, indicating that the PWWP domain is a
chromatin reader module for H3K36me2/3 in general (Vezzoli
et al., 2010; Okuda et al., 2014). H3K36me2 marks are found
in active gene promoters and are introduced by histone methyl
transferases (HMTs) like ASH1L and NSD2 (Kuo et al., 2011; Zhu
et al., 2016). When transcription is ongoing, further methylation
on an H3K36me2 mark in the gene body region occurs to
produce H3K36me3 by another HMT termed SETD2 complexed
with elongating RNAP2 (Wagner and Carpenter, 2012). This
H3K36me3 modification highlights transcribed regions and is
required for efficient DNA damage response (Mar et al., 2017).
Therefore, heterozygous loss of SETD2, which leads to blunt
DNA damage response against chemotherapy, is often found in
relapsed leukemia (Mullighan et al., 2011; Mar et al., 2014; Xiao
et al., 2016). SETD2 was recently reported to physically interact
with MLL fusion proteins and may also be implicated in the
efficient targeting of MLL fusion proteins to the target promoters
(Skucha et al., 2018).
UNMETHYLATED CG DNA SEQUENCE
Unmethylated CG DNA sequence, which are specifically
recognized by the CXXC domain, are enriched in gene
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
promoters, and are linked to transcription initiation (Cedar and
Bergman, 2009; Bird, 2011). If the cytosine of CpGs is methylated,
the CXXC domain no longer binds to the CG sequence
(Allen et al., 2006). Unmethylated CpGs are an epigenetic
mark of non-silenced promoters because methylation of CpGs
in the promoter are associated with transcriptional silencing.
Through the PWWP and CXXC domains, the MLL fusion
complex targets transcriptionally-active CpG-rich promoters
(Figure 3D). During embryogenesis, MLL maintains segmentspecific
expression of HOX genes (Yu et al., 1995). HOX
genes are called “cellular memory” genes as their positionspecific
expression patterns are maintained during development
(Deschamps and van Nes, 2005; Wang et al., 2009). MLL is
not required for initial activation of HOX gene expression, but
is required for the maintenance of HOX gene expression (Yu
et al., 1998), indicating that MLL is involved in maintaining an
established expression pattern, rather than in determining the
expression pattern itself. Therefore, it is likely that MLL targets
CpG-rich promoters which were previously transcriptionallyactive
in the maternal cell and re-activates transcription in
daughter cells to maintain the HOX gene expression patterns.
ADDITIONAL MECHANISMS OF TARGET
RECOGNITION BY MLL FUSION PROTEINS
Resent research showed that the target chromatin of MLL
fusion proteins is not confined to the promoter region.
Localization of MLL fusion proteins spreads into the gene
body at some MLL target genes, which are often hypomethylated
and highly transcribed (Kerry et al., 2017). It has
been suggested that this aberrant localization is implicated in
disease progression. Moreover, in some genes MLL-ENL localizes
near the transcription end site and activates gene expression
predominantly at transcription elongation levels (Garcia-Cuellar
et al., 2016). These results suggest that MLL fusion proteins
can be involved in multiple facets of gene activation through
binding outside of the promoter region even though their
primary targets are CpG-rich promoter regions. Moreover, the
structure juxtaposed to the CXXC domain of MLL has been
shown to associate with the PAF1 complex, which is thought
to bind elongating RNAP2 (Milne et al., 2010; Muntean et al.,
2010). PAF1 association may be involved in the fine-tuning of
target recognition and/or noncanonical targeting mechanisms
mentioned above.
SL1-MEDIATED TRANSCRIPTIONAL
ACTIVATION BY MLL FUSION PROTEINS
While MLL fusion proteins target previously-active CpG-rich
promoters through their MLL portions, the fusion partner
portion confers the ability to activate transcription. The
minimum domains within the fusion partner portion required
for transformation have been identified for MLL-ENL and MLLAF5Q31
(Slany et al., 1998; Yokoyama et al., 2010; Okuda
et al., 2015). The ANC1 homology domain (AHD) of ENL
and the C-terminal homology domain (CHD) of AF5Q31
are responsible for Hoxa9 transcriptional activation and for
the transformation of myeloid progenitors (Figure 4A). These
domains are the binding platforms for AF4, suggesting that
aberrant recruitment of AF4 to MLL target promoters is essential
for MLL fusion-dependent transformation. Therefore, functional
modules retained within the AF4 portion were thought to be
responsible for transformation. An artificial construct in which
the pSER domain of AF4 is tethered to the MTM activated Hoxa9
expression and immortalized myeloid progenitors (Figure 4B),
indicated that recruitment of the SL1 complex to MLL target
promoters is necessary and sufficient for transformation. The
minimum structure required for transformation was the region
encompassing the SDE motif and the NKW motif, which recruit
SL1 and activate transcription (Okuda et al., 2015, 2016). These
results suggest that the MLL fusion proteins transform myeloid
progenitors via SL1-mediated transcriptional activation through
AF4. Surprisingly, other functional modules such as the Nterminal
homology domain (NHD), which recruits P-TEFb,
and the AF4/LAF4/FMR2 homology (ALF) domain (Nilson
et al., 1997), which recruits ELL, were dispensable despite the
much anticipated significance of these elongation factors. Taken
together, these observations suggest that the major role of MLL
fusion proteins in leukemic transformation is not to activate
transcription elongation but to activate transcription initiation
via SL1. Similarly, the DLXLS motif, which recruits Mediator,
was also dispensable for transformation (Okuda et al., 2016),
suggesting that direct recruitment of Mediator by the MLL
fusion complex appears not critical either. Hence, SL1-mediated
transcriptional activation by RNAP2 is the rate-limiting step for
MLL fusion-dependent gene activation (Figures 5A,B).
MAINTENANCE OF MLL
FUSION-DEPENDENT TRANSCRIPTION
BY DOT1L
Transcriptional maintenance is also required for MLL fusionmediated
leukemogenesis. MLL target genes are prone to gene
silencing by transcriptional repressors such as SIRT1 histone
deacetylase (Chen C. W. et al., 2015). To maintain gene
expression, MLL fusion proteins utilize the DOT1L HMT.
DOT1L is an epigenetic modifier that produces mono-, di-,
and tri-methylated histone H3 lysine 79 marks (H3K79me1/2/3)
(Feng et al., 2002; Jones et al., 2008). DOT1L forms a complex
with AF10 family proteins (AF10/AF17) and ENL family proteins
(ENL/AF9) (Okada et al., 2005; Mueller et al., 2007; Mohan
et al., 2010) (Figure 4A). The association of DOT1L with AF10
increases DOT1L HMT activity (Deshpande et al., 2014). The
AF10 family genes also form a MLL fusion gene to cause leukemia
(DiMartino et al., 2002). MLL-ENL and MLL-AF10 directly
recruit DOT1L to the target promoters, suggesting that aberrant
DOT1L recruitment contributes to leukemogenesis (Okuda et al.,
2017). Moreover, MLL fusion-transformed myeloid progenitors
lose their clonogenicity upon acute loss of the Dot1l gene (Chang
et al., 2010; Bernt et al., 2011; Jo et al., 2011; Nguyen et al.,
2011; Chen et al., 2013). These observations indicated that
the continuous presence of DOT1L is required for leukemic
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
FIGURE 4 | Structural requirements of MLL fusion proteins for leukemic transformation. (A) Schematic representation of the structures of various MLL fusion
constructs. MLL fusion proteins recruit AF4 and ENL through different mechanisms to immortalize hematopoietic progenitors. The minimal construct of MLL-ENL
which immortalizes hematopoietic progenitors (MTM-ENL’) is composed of MTM and the AHD of ENL, while that of MLL-AF5Q31(MTM-AF5-4) is composed of MTM
and the CHD of AF5Q31, indicating that recruitment of AF4 to the MLL target chromatin confers transforming activity. By contrast, the minimal transforming construct
of MLL-AF10 (MTM-TRX2-AF10’) is composed of MTM, the TRX2 domain, and the OMLZ domain of AF10, indicating that MLL-AF10 requires the TRX2 domain for
AF4 recruitment and the OMLZ domain for ENL recruitment. In concordance with this notion, the MTM-TRX2-DOT1L construct immortalizes hematopoietic
progenitors whereas deletion of the ENL binding domain (MISD) results in loss of transformation. Dotted lines indicate protein-protein interaction. Associated
properties of each construct, such as the ability to immortalize myeloid progenitors, and the binding abilities to AF4, DOT1L, and ENL are shown on the right.
Immortalizing ability and AF4 binding ability through the TRX2 domain are highlighted in blue and red, respectively. MISD: minimum interaction site for DOT1L. One
MISD, located at the residues 628–653, was omitted because of its very weak affinity (Kuntimaddi et al., 2015). N.A., not applicable. (B) Schematic representation of
the structures of various MTM-AF4 fusion constructs. MTM-AF4 fusion proteins recruit SL1 through the SDE motif and activate transcription through the NKW motif to
immortalize hematopoietic progenitors. Binding modules for P-TEFb or ELL did not confer transforming ability. Associated properties of each construct, including the
ability to immortalize myeloid progenitors, binding abilities to SL1, MED26, P-TEFb, and ELL are shown on the right.
transformation and led to the development of a DOT1L HMT
inhibitor for the treatment of MLL-r leukemia (Daigle et al.,
2011). EPZ-5676 (Pinometostat), a potent DOT1L inhibitor,
showed remarkable efficacy in rodent xenograft models (Daigle
et al., 2013), confirming that DOT1L-dependent transcriptional
maintenance is required for MLL fusion proteins. However, the
mode of DOT1L recruitment is somewhat controversial. Some
studies suggest that AF4 proteins form a stable complex with
DOT1L (Bitoun et al., 2007; Lin et al., 2016). Our biochemical
data suggest that AF4 family proteins do not directly associate
with DOT1L (Yokoyama et al., 2010). AF4 family proteins
and DOT1L bind to the AHD of ENL family proteins in a
mutually exclusive manner (Mueller et al., 2007; Yokoyama et al.,
2010; Okuda et al., 2017). Structural studies showed that similar
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
FIGURE 5 | Models of MLL fusion-dependent gene activation. Working models of gene activation by MLL-ENL (A), MLL-AF4/AF5Q31 (B), and MLL-AF10 (C) are
shown. (A) MLL-ENL associates with the DOT1L complex on the target chromatin. Because AF4 binds ENL with higher affinity than DOT1L, MLL-ENL preferentially
forms a complex with AEP. AEP components recruit SL1 to activate transcription while the DOT1L complex maintains transcriptionally-active chromatin by methylating
histone H3 lysine 79 to repel SIRT1 transcriptional repressor. (B) MLL-AF4/AF5q31 recruits AF4 proteins, which subsequently bind to ENL tethered to chromatin
containing histone H3 K9/18/27ac marks. AEP activates transcription via SL1. (C) MLL-AF10 forms a complex with DOT1L and ENL on the target chromatin. AF4
proteins are recruited via the TRX2 domain, which then bind to ENL tethered to chromatin containing histone H3 K9/18/27ac marks to form AEP.
hydrophobic motifs in DOT1L and AF4 bind to the same groovelike
structure in AHD (Leach et al., 2013; Shen et al., 2013;
Kuntimaddi et al., 2015). Therefore, AF4-ENL association and
DOT1L-ENL association via AHD should be mutually exclusive.
I postulate that AF4 proteins cannot form a complex with
DOT1L because of this structural restraint, but do not exclude
the possibility that AF4 and DOT1L can be tethered in alternative
indirect manners.
MECHANISMS OF GENE ACTIVATION BY
MLL-ENL AND MLL-AF10
The minimum domain structure of the MLL portion required
for transformation by MLL-AF10 differs from that required by
MLL-ENL (Figure 4A). As for MLL-ENL, the MTM and the
AHD, which are sufficient to recruit both AF4 and DOT1L to
the MLL target genes, confer transforming ability (Okuda et al.,
2014, 2015). In contrast, the MTM fused with the OMLZ domain
of AF10 (MTM-AF10′), which recruits DOT1L but not AF4,
exhibited relatively high Hoxa9 expression in first round colonies
but could not maintain its expression for a longer period and
was unable to immortalize myeloid progenitors (Okuda et al.,
2017). This indicated that DOT1L recruitment to the MLL-target
promoter upregulates target gene expression insufficiently for
immortalization. However, incorporation of the TRX2 domain
of MLL into this MTM-AF10′ fusion construct (MTM-TRX2AF10′)
resulted in full transformation, indicating that the TRX2
domain confers additional functions to achieve full leukemic
transformation. Given that one function retained by the AHD,
but missing from the OMLZ domain, is the ability to recruit
AF4 family proteins, we examined whether the TRX2 domain
associates with AF4 family proteins on chromatin. To this end, we
used the fractionation-assisted chromatin immunoprecipitation
(fanChIP) method (Okuda et al., 2014), which enables us to
capture protein complexes bound to chromatin. Indeed, the
TRX2 domain associated with AF4 proteins (Okuda et al., 2017).
Thus, MLL-AF10 recruits AF4 and DOT1L through the TRX2
domain and the OMLZ domain, respectively, to immortalize
hematopoietic progenitors (Figure 5C).
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
Moreover, artificial MLL-DOT1L constructs exhibited similar
structural requirement (Figure 4A). An artificial construct in
which the MTM is fused to the entire DOT1L coding sequence
(MTM-DOT1L), exhibited the same compromised transforming
property as the MTM-AF10′ construct, while incorporation
of the TRX2 domain (MTM-TRX2-DOT1L) conferred full
transforming ability. These results confirmed that recruitment
of both AF4 and DOT1L is required for MLL-AF10-dependent
transformation. Deletion of the ENL binding domains (MISD:
the minimum interaction site of DOT1L) from the MTM-TRX2DOT1L
construct (MTM-TRX2-DOT1L dMISD) resulted in loss
of transformation, indicating that the ENL-DOT1L association
is required for MLL-AF10-dependent transformation. This result
contradicts a previous report which showed that an artificial
fusion of MLL and the HMT domain of DOT1L (MLLDOT1L
HMT), lacking the ENL binding domains, transformed
myeloid progenitors in a similar setting (Okada et al., 2005).
However, we consistently obtain no transformation readout
using this MLL-DOT1L HMT construct (Yokoyama et al.,
2010), supporting our conclusion that simply recruiting DOT1L
HMT activity to the MLL target chromatin is insufficient for
leukemic transformation. Based on these results, I propose a
model in which MLL-AF10 promotes AEP formation on nearby
chromatin through AF4 recruitment by the TRX2 domain and
ENL recruitment by DOT1L (Figure 5C). Thus, both MLL-ENL
and MLL-AF10 appear to activate transcription in an AEP/SL1dependent
manner.
THE ROLE OF TRX2 DOMAIN OF MLL
It is unclear how AF4 proteins associate with the TRX2 domain
of MLL. Interaction between MLL and AF4 was not detected
in conventional IP analysis (Yokoyama et al., 2010). Thus
far, this association has only been detected in the chromatin
context (Okuda et al., 2017). It is possible that some other
chromatin-bound factors mediate the interaction between MLL
and AF4 through the TRX2 domain. It has been reported that
the region containing the TRX2 domain also associates with
SHARP1, which may be involved in interaction between MLL
and AF4 (Numata et al., 2018). Although the MLL 5′ portion
retains the TRX2 domain, it cannot activate transcription of
Hoxa9 and transform myeloid progenitors without its fusion
partner portion (Lavau et al., 1997; Slany et al., 1998; Okuda
et al., 2017), indicating that AF4 bound with the TRX2
domain is transcriptionally inactive and needs to form an AEP
complex with ENL on nearby chromatin to become functional
(Figure 5C). Supporting this hypothesis, ENL knockdown in
MLL-AF10-transformed cells resulted in loss of colony forming
ability (Okuda et al., 2017).
MECHANISM OF TARGET RECOGNITION
BY THE DOT1L COMPLEX
Whether DOT1L must be directly recruited by MLL fusion
proteins also remains unclear. MLL-ENL directly associates
with DOT1L and AF4 through the AHD (Mueller et al., 2007;
Yokoyama et al., 2010; Leach et al., 2013; Kuntimaddi et al.,
2015). ChIP-seq analysis of HB1119 cells (which express MLLENL)
showed remarkable overlap of the ChIP-signals of MLLENL,
AF4, and DOT1L (Okuda et al., 2017). However, it
is unclear whether MLL-AF4 directly recruits DOT1L to the
target chromatin. Several reports have demonstrated interaction
between DOT1L and MLL-AF4 or AF4 by immunoprecipitation
(Bitoun et al., 2007; Lin et al., 2016). Moreover, H3K79me2
marks produced by DOT1L are associated with MLL-AF4 target
genes (Krivtsov et al., 2008; Kerry et al., 2017), suggesting a
mechanism that MLL-AF4 might directly recruit DOT1L to its
target chromatin. However, our biochemical data demonstrated
that AF4 proteins do not form a stable complex with DOT1L
(Yokoyama et al., 2010; Okuda et al., 2017). This indicates that
DOT1L may autonomously target similar chromatin to that
targeted by MLL and AEP without the help of MLL fusion
proteins. The DOT1L complex retains its own chromatin binding
modules. AF10 family proteins specifically bind to unmodified
histone H3 lysine 27 (H3K27 me0) through their PHD fingerZn
knuckle-PHD finger (PZP) domain (Chen S. et al., 2015)
(Figure 4A). The YEATS domain of ENL binds to acetylated
histone H3 lysine 9/18/27 (Li et al., 2014; Erb et al., 2017;
Wan et al., 2017). With those chromatin reader modules, the
DOT1L complex possibly binds to its target chromatin by
itself. Because AEP and the DOT1L complex both share the
ENL family proteins as a component, they should target the
same chromatin containing acetylated histone H3 K9/18/27.
Consistent with this hypothesis, AEP and the DOT1L complex
colocalized at the promoter-proximal regions of ENL target genes
in HEK293T cells (Okuda et al., 2017). Binding affinity of ENL
family proteins to AF4 family proteins is stronger than that
to DOT1L in vitro (Leach et al., 2013). Therefore, the DOT1L
complex likely provides ENL family proteins to AF4 family
proteins nearby. As such, the DOT1L complex promotes the
chromatin association of AEP. AEP/SL1-mediated transcription
may in turn stimulate recruitment of the DOT1L complex in
a feedback loop mechanism as mono-ubiquitination of histone
H2B, which is coupled with transcription, stimulates DOT1Ldependent
methylation activity of Histone H3 (McGinty et al.,
2008). Such interplay between AEP and the DOT1L complex
appears to be present and needs to be investigated in more detail
in the future.
DIRECT RECRUITMENT OF AEP NOT
DOT1L IS REQUIRED FOR LEUKEMIC
TRANSFORMATION BY MLL FUSION
PROTEINS
Structure/function analysis data using the myeloid progenitor
transformation assay indicate that modules that recruit AEP,
but not DOT1L, are necessary and sufficient for transformation
by MLL fusion proteins (Figure 4A). For instance, MLL fused
with the CHD of AF5Q31, which binds to AF4 but not DOT1L,
transformed myeloid progenitors (Okuda et al., 2015, 2017). The
MLL-Af4 fusion gene, in which the human MLL gene is fused
to murine Af4 gene, was shown to transform hematopoietic
progenitors to develop leukemia in vivo (Lin et al., 2016). An
artificial construct in which MLL is fused to the CHD portion
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Yokoyama SL1 Mediates RNAP2-Dependent Transcription
of murine AF4 successfully induced leukemia in vivo (Lin et al.,
2017). These observations favor the model that direct recruitment
of AF4 family proteins but not DOT1L is the critical step for
MLL fusion-dependent gene activation, and that the DOT1L
complex targets to similar chromatin autonomously to maintain
transcription (Figure 5B). Formation of the MLL fusion/MENIN
complex can be inhibited by a small compound (MI-2, MI-
2-2, MI-463, MI-503) (Grembecka et al., 2012; Shi et al.,
2012). Simultaneous inhibition of MENIN-MLL interaction and
DOT1L HMT activity synergistically induces differentiation of
MLL fusion-associated leukemia cells (Dafflon et al., 2017; Okuda
et al., 2017), supporting the notion that the MLL fusion complex
and the DOT1L complex collaborate to induce leukemia.
HOW DOES SL1 ACTIVATE
RNAP2-DEPENDENT TRANSCRIPTION?
Multiple lines of evidence support the notion that SL1 is
involved in AEP-dependent gene activation. For example,
genome-wide ChIP-seq analysis demonstrated that TAF1C colocalizes
with AF4 and RNAP2 at transcription start sites
(Okuda et al., 2015, 2017). Taf1c knockdown causes downregulation
of AEP-target genes in mouse embryonic fibroblasts
(Okuda et al., 2015). Moreover, the pSER domain, which is
the binding platform for SL1, can be functionally replaced by
a transcriptional activation domain for RNAP2. However, the
precise mechanism by which SL1 activates RNAP2-dependent
transcription is still unknown. A GAL4-pSER fusion protein
activates RNAP2-dependent transcription on an artificial GAL4responsive
promoter containing a TATA box. This activation can
be abolished if the TATA box is removed, indicating that the pSER
domain promotes loading of TBP onto the TATA box in the form
of SL1. After loading of TBP, SL1 must be dismantled and then
TAF1B needs to be replaced by TFIIB, which share structural and
functional similarities (Naidu et al., 2011). This leads RNAP2 to
form an RNAP2-PIC (Figure 2C). The NKW motif of the pSER
domain is required for transactivation and transformation and
therefore is expected to play an important role. I speculate that
the NKW motif binds to SL1 to induce conformational changes
that facilitate either TBP loading and/or TFIIB exchange.
WHY DOES MLL FUSION PREFER AEP
COMPONENTS AS ITS FUSION PARTNER?
It is odd that MLL predominantly prefers AEP components
as its fusion partner. AEP/SL1-dependent gene activation
seems much less potent compared to that of other activation
domains that recruit TFIID (Okuda et al., 2015). Yet, AEP
components are preferentially chosen by MLL. This suggests
that AEP/SL1-dependent transcriptional activation has some
advantage over TFIID-dependent transcriptional activation.
Transcription oscillates during the cell cycle (Gottesfeld and
Forbes, 1997; Liu et al., 2017). The metaphase Cyclin/CDK
complex phosphorylates SL1, which hinders the interaction
between SL1 and UBF, to shut off rRNA transcription during
metaphase (Heix et al., 1998). UBF is also inactivated by
phosphorylation during metaphase (Klein and Grummt, 1999).
Interestingly, SL1 is re-activated in early G1 while UBF
remains inactive, suggesting a role for SL1 in addition to
rRNA transcription. Therefore, it is possible that AEP/SL1dependent
RNAP2 activation starts in early G1 phase. Unlike
many sequence-specific transcription factors, MLL is tethered
to chromatin during mitosis, setting the stage for efficient
transcription in the next early G1 phase (Blobel et al., 2009).
Perhaps transcription of those MLL target genes starts as soon as
the next G1 begins, potentially explaining the preference for AEP
components as MLL fusion partners. Such biological property
may be suited for persistent transcriptional activation of HSC
programs genes by the MLL/AEP axis. Given the important
roles of SL1 in RNAP1-dependent transcription, gene knockout
technologies are not applicable to address these questions.
Emerging rapid degradation technology used in the studies of
essential factors for viability (Baptista et al., 2017; Warfield et al.,
2017) may need to be applied for SL1 components to provide
further insights. Because MLL fusion proteins heavily rely on
AEP/SL1-dependent gene activation, compounds that inhibit this
gene activation process could be used as drugs to treat MLL-r
leukemia patients.
CONCLUDING REMARKS
In conclusion, accumulating evidence indicates that RNAP2dependent
transcription mediated by SL1 is a central mechanism
used by MLL fusion proteins. However, much of its molecular
mechanism remains undocumented and needs to be investigated.
Hopefully, precise mechanisms of transcriptional activation by
AEP and SL1 will be revealed in greater detail over the next
decade.
AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and
has approved it for publication.
ACKNOWLEDGMENTS
This study was supported by a Japan Society for the Promotion of
Science (JSPS) KAKENHI grant (16H05337) to AY.
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