Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation
. However, redundant functions of the H3K4 ...SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation
. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the ...functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.
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•The NSL but not the MSL complex is essential for the proliferation of human cells•The NSL complex places H4K5ac and H4K8ac at TSSs, promoting transcription initiation•H4K16ac is a highly abundant modification placed by KAT8 as part of the MSL complex•H4K16ac marks open chromatin but does not affect global chromatin accessibility
Radzisheuskaya et al. report that histone acetyltransferase KAT8 switches functions depending on associated proteins. It catalyzes promoter-associated H4K5 and H4K8 acetylation as part of the NSL complex, which is required for the activation of essential genes. As part of the MSL complex, it places H4K16 acetylation marking all open chromatin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we ...investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections
. A four-strained ...consortium of commensal bacteria that contains Blautia producta BP
can reverse antibiotic-induced susceptibility to VRE infection
. Here we show that BP
reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BP
and L. lactis in vitro, only BP
colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BP
lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect ...their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N- and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In ...embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.
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•Interactome analysis highlights Tex10 as a binding partner of Sox2•Tex10 is required for maintenance and establishment of pluripotency•Tex10 regulates super-enhancer activity via epigenetic modifications•Sox2 targets Tex10 to ESC-specific super-enhancers
Using Sox2-centered interactome analysis, Ding et al. identify Tex10 as an evolutionarily conserved key pluripotency factor that plays a critical role in ESC self-renewal and pluripotency, early embryo development, and somatic cell reprogramming via epigenetic regulation of super-enhancer activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Oncogenic alterations to DNA are not transforming in all cellular contexts
. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a ...major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails
. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core ...pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.
Efficient treatment of acute myeloid leukemia (AML) patients remains a challenge despite recent therapeutic advances. Here, using a CRISPRi screen targeting chromatin factors, we identified the ...nucleosome‐remodeling factor (NURF) subunit BPTF as an essential regulator of AML cell survival. We demonstrate that BPTF forms an alternative NURF chromatin remodeling complex with SMARCA5 and BAP18, which regulates the accessibility of a large set of insulator regions in leukemic cells. This ensures efficient CTCF binding and boundary formation between topologically associated domains that is essential for maintaining the leukemic transcriptional programs. We also demonstrate that the well‐studied PHD2‐BROMO chromatin reader domains of BPTF, while contributing to complex recruitment to chromatin, are dispensable for leukemic cell growth. Taken together, our results uncover how the alternative NURF complex contributes to leukemia and provide a rationale for its targeting in AML.
Synopsis
Chromatin regulators play a crucial role in cancer, including acute myeloid leukemia (AML). Here, a screen for new vulnerabilities in MLL‐rearranged AML identifies an alternative NURF chromatin remodelling complex that maintains leukemic transcriptional programs.
CRISPRi screen in AML cells identifies BPTF as an essential regulator of cell growth.
BPTF interacts with SMARCA5 and BAP18 to form an alternative NURF complex in AML cells.
The NURF complex controls insulator accessibility, CTCF binding, and TAD boundary formation.
PHD2 and BROMO domains of BPTF are not required for leukemic cell growth.
Chromatin remodeling factors BPTF, SMARCA5 and BAP18 form a complex that regulates insulator accessibility and higher‐order genome organization in acute myeloid leukemia.
Biological functions of many proteins are governed by post-translational modifications (PTMs). In particular, the rich PTM complement in histones controls the gene expression and chromatin structure ...with major health implications via a combinatoric language. Deciphering that “histone code” is the great challenge for proteomics given an astounding number of possible proteoforms, including isomers with different PTM positions. These must be disentangled on the top- or middle-down level to preserve the key PTM connectivity, which condensed-phase separations failed to achieve. We reported the capability of ion mobility spectrometry (IMS) methods to resolve such isomers for model histone tails. Here, we advance to biological samples, showing middle-down analyses of histones from mouse embryonic stem cells via online chromatography to fractionate proteoforms with distinct PTM sets, differential or field asymmetric waveform IMS (FAIMS) to resolve the isomers, and Orbitrap mass spectrometry with electron transfer dissociation to identify the resolved species.
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IJS, KILJ, NUK, PNG, UL, UM
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