Zhanyun Tang

RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation

Histone modifications regulate chromatin-dependent processes, yet the mechanisms by which they contribute to specific outcomes remain unclear. H3K4me3 is a prominent histone mark that is associated with active genes and promotes transcription through interactions with effector proteins that include initiation factor TFIID. We demonstrate that H3K4me3-TAF3 interactions direct global TFIID recruitment to active genes, some of which are p53 targets. Further analyses show that (1) H3K4me3 enhances p53-dependent transcription by stimulating preinitiation complex (PIC) formation; (2) H3K4me3, through TAF3 interactions, can act either independently or cooperatively with the TATA box to direct PIC formation and transcription; and (3) H3K4me3-TAF3/TFIID interactions regulate gene-selective functions of p53 in response to genotoxic stress. Our findings indicate a mechanism by which H3K4me3 directs PIC assembly for the rapid induction of specific p53 target genes.

A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.

Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters

Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.

RNA polymerase II–associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II

“Please release me, let me go.” RNA polymerase II (Pol II) is the principal protein complex required for gene transcription in metazoan cells. Many genes have a “paused” Pol II near their promoters, waiting to be released so they can start messenger RNA synthesis. Yu et al. show that Pol II–associated factor 1 (PAF1) plays a central role in regulating the activation of these paused Pol II complexes. The positive transcription elongation factor b helps recruit PAF1 to the paused Pol II. This facilitates the phosphorylation of Pol II on its C-terminal domain, freeing it to start transcription in earnest. Science, this issue p. 1383 RNA polymerase is paused near the promoters of many genes, and Pol II–associated factor 1 plays a critical role in its release. Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity–inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II–associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.

USF1 and hSET1A Mediated Epigenetic Modifications Regulate Lineage Differentiation and HoxB4 Transcription

The interplay between polycomb and trithorax complexes has been implicated in embryonic stem cell (ESC) self-renewal and differentiation. It has been shown recently that WRD5 and Dpy-30, specific components of the SET1/MLL protein complexes, play important roles during ESC self-renewal and differentiation of neural lineages. However, not much is known about how and where specific trithorax complexes are targeted to genes involved in self-renewal or lineage-specification. Here, we report that the recruitment of the hSET1A histone H3K4 methyltransferase (HMT) complex by transcription factor USF1 is required for mesoderm specification and lineage differentiation. In undifferentiated ESCs, USF1 maintains hematopoietic stem/progenitor cell (HS/PC) associated bivalent chromatin domains and differentiation potential. Furthermore, USF1 directed recruitment of the hSET1A complex to the HoxB4 promoter governs the transcriptional activation of HoxB4 gene and regulates the formation of early hematopoietic cell populations. Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation. We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation. Finally, ectopic expression of USF1 in ESCs promotes mesoderm differentiation and enforces the endothelial-to-hematopoietic transition by inducing hematopoietic-associated transcription factors, HoxB4 and TAL1. Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

Histone H3K27 Trimethylation Inhibits H3 Binding and Function of SET1-Like H3K4 Methyltransferase Complexes

ABSTRACT Trimethylated histone H3 lysine 4 (H3K4) and H3K27 generally mark transcriptionally active and repressive chromatins, respectively. In most cell types, these two modifications are mutually exclusive, and this segregation is crucial for the regulation of gene expression. However, how this anticorrelation is achieved has not been fully understood. Here, we show that removal of the H3K27 trimethyl mark facilitates recruitment of SET1-like H3K4 methyltransferase complexes to their target genes by eliciting a novel interaction between histone H3 and two common subunits, WDR5 and RBBP5, of SET1-like complexes. Consistent with this result, H3K27 trimethylation destabilizes interactions of H3 with SET1-like complexes and antagonizes their ability to carry out H3K4 trimethylation of peptide (H3 residues 1 to 36), histone octamer, and mononucleosome substrates. Altogether, our studies reveal that H3K27 trimethylation of histone H3 represses a previously unrecognized interaction between H3 and SET1-like complexes. This provides an important mechanism that directs the anticorrelation between H3K4 and H3K27 trimethylation.

Regulation of transcription by the MLL2 complex and MLL complex-associated AKAP95

Although histone H3 Lys4 (H3K4) methylation is widely associated with gene activation, direct evidence for its causal role in transcription, through specific MLL family members, is scarce. Here we have purified a human MLL2 (Kmt2b) complex that is highly active in H3K4 methylation and chromatin transcription in a cell-free system. This effect requires S-adenosyl methionine and intact H3K4, thus establishing a direct and causal role for MLL2-mediated H3K4 methylation in transcription. We also show that human AKAP95, a chromatin-associated protein, physically and functionally associates with MLL complexes and directly enhances their methyltransferase activity. Ectopic AKAP95 stimulates expression of a chromosomal reporter gene in synergy with MLL1 or MLL2, whereas AKAP95 depletion impairs retinoic acid–mediated gene induction in embryonic stem cells. These results demonstrate an important role for AKAP95 in regulating histone methylation and gene expression, particularly during cell-fate transitions.

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Significance This article applies a range of protein ligation methods at the level of chromatin to understand the cross-talk mechanism between the well-established biomedical target the human Dot1 (hDot1L) methyltransferase and the ubiquitylation of H2B at lysine 120. Through a systematic structure–activity relationship study of the ubiquitin surface in regard to hDot1L-mediated H3K79 methylation and further investigation with precisely engineered chromatin substrates, a functional hotspot within ubiquitin was identified that is essential to the stimulation of hDot1L activity. More broadly, this work shows how chemical synthesis approaches can be used to precisely tailor protein posttranslational modifications to afford mechanistic insights that would be impossible by other methods. Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure–activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub–induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.

JMJD1C is required for the survival of acute myeloid leukemia by functioning as a coactivator for key transcription factors

RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program. JMJD1C is directly recruited by RUNX1-RUNX1T1 to its target genes and regulates their expression by maintaining low H3K9 dimethyl (H3K9me2) levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for RUNX1-RUNX1T1s ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors.