RNA damage compartmentalization by DHX9 stress granules.

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

MSL2 ensures biallelic gene expression in mammals.

In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter-enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.

COX17 acetylation via MOF-KANSL complex promotes mitochondrial integrity and function.

Reversible acetylation of mitochondrial proteins is a regulatory mechanism central to adaptive metabolic responses. Yet, how such functionally relevant protein acetylation is achieved remains unexplored. Here we reveal an unprecedented role of the MYST family lysine acetyltransferase MOF in energy metabolism via mitochondrial protein acetylation. Loss of MOF-KANSL complex members leads to mitochondrial defects including fragmentation, reduced cristae density and impaired mitochondrial electron transport chain complex IV integrity in primary mouse embryonic fibroblasts. We demonstrate COX17, a complex IV assembly factor, as a bona fide acetylation target of MOF. Loss of COX17 or expression of its non-acetylatable mutant phenocopies the mitochondrial defects observed upon MOF depletion. The acetylation-mimetic COX17 rescues these defects and maintains complex IV activity even in the absence of MOF, suggesting an activatory role of mitochondrial electron transport chain protein acetylation. Fibroblasts from patients with MOF syndrome who have intellectual disability also revealed respiratory defects that could be restored by alternative oxidase, acetylation-mimetic COX17 or mitochondrially targeted MOF. Overall, our findings highlight the critical role of MOF-KANSL complex in mitochondrial physiology and provide new insights into MOF syndrome.

Transcriptional regulation by the NSL complex enables diversification of IFT functions in ciliated versus nonciliated cells.

Members of the NSL histone acetyltransferase complex are involved in multiorgan developmental syndromes. While the NSL complex is known for its importance in early development, its role in fully differentiated cells remains enigmatic. Using a kidney-specific model, we discovered that deletion of NSL complex members KANSL2 or KANSL3 in postmitotic podocytes led to catastrophic kidney dysfunction. Systematic comparison of two primary differentiated cell types reveals the NSL complex as a master regulator of intraciliary transport genes in both dividing and nondividing cells. NSL complex ablation led to loss of cilia and impaired sonic hedgehog pathway in ciliated fibroblasts. By contrast, nonciliated podocytes responded with altered microtubule dynamics and obliterated podocyte functions. Finally, overexpression of wild-type but not a double zinc finger (ZF-ZF) domain mutant of KANSL2 rescued the transcriptional defects, revealing a critical function of this domain in NSL complex assembly and function. Thus, the NSL complex exhibits bifurcation of functions to enable diversity of specialized outcomes in differentiated cells.

The NSL complex is required for piRNA production from telomeric clusters.

The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposons HeT-A, TAHRE, and TART Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. Our findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline.

Asifa Akhtar.

Q&A with Asifa Akhtar who studies histone acetylation and genome regulation.

Modulation of cellular processes by histone and non-histone protein acetylation.

Lysine acetylation is a widespread and versatile protein post-translational modification. Lysine acetyltransferases and lysine deacetylases catalyse the addition or removal, respectively, of acetyl groups at both histone and non-histone targets. In this Review, we discuss several features of acetylation and deacetylation, including their diversity of targets, rapid turnover, exquisite sensitivity to the concentrations of the cofactors acetyl-CoA, acyl-CoA and NAD+, and tight interplay with metabolism. Histone acetylation and non-histone protein acetylation influence a myriad of cellular and physiological processes, including transcription, phase separation, autophagy, mitosis, differentiation and neural function. The activity of lysine acetyltransferases and lysine deacetylases can, in turn, be regulated by metabolic states, diet and specific small molecules. Histone acetylation has also recently been shown to mediate cellular memory. These features enable acetylation to integrate the cellular state with transcriptional output and cell-fate decisions.

Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice.

Noncommunicable diseases (NCDs) account for over 70% of deaths world-wide. Previous work has linked NCDs such as type 2 diabetes (T2D) to disruption of chromatin regulators. However, the exact molecular origins of these chronic conditions remain elusive. Here, we identify the H4 lysine 16 acetyltransferase MOF as a critical regulator of central carbon metabolism. High-throughput metabolomics unveil a systemic amino acid and carbohydrate imbalance in Mof deficient mice, manifesting in T2D predisposition. Oral glucose tolerance testing (OGTT) reveals defects in glucose assimilation and insulin secretion in these animals. Furthermore, Mof deficient mice are resistant to diet-induced fat gain due to defects in glucose uptake in adipose tissue. MOF-mediated H4K16ac deposition controls expression of the master regulator of glucose metabolism, Pparg and the entire downstream transcriptional network. Glucose uptake and lipid storage can be reconstituted in MOF-depleted adipocytes in vitro by ectopic Glut4 expression, PPARγ agonist thiazolidinedione (TZD) treatment or SIRT1 inhibition. Hence, chronic imbalance in H4K16ac promotes a destabilisation of metabolism triggering the development of a metabolic disorder, and its maintenance provides an unprecedented regulatory epigenetic mechanism controlling diet-induced obesity.

Differential H4K16ac levels ensure a balance between quiescence and activation in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are able to reconstitute the bone marrow while retaining their self-renewal property. Individual HSCs demonstrate heterogeneity in their repopulating capacities. Here, we found that the levels of the histone acetyltransferase MOF (males absent on the first) and its target modification histone H4 lysine 16 acetylation are heterogeneous among HSCs and influence their proliferation capacities. The increased proliferative capacities of MOF-depleted cells are linked to their expression of CD93. The CD93+ HSC subpopulation simultaneously shows transcriptional features of quiescent HSCs and functional features of active HSCs. CD93+ HSCs were expanded and exhibited an enhanced proliferative advantage in Mof +/- animals reminiscent of a premalignant state. Accordingly, low MOF and high CD93 levels correlate with poor survival and increased proliferation capacity in leukemia. Collectively, our study indicates H4K16ac as an important determinant for HSC heterogeneity, which is linked to the onset of monocytic disorders.

Distinct mechanisms mediate X chromosome dosage compensation in Anopheles and Drosophila.

Sex chromosomes induce potentially deleterious gene expression imbalances that are frequently corrected by dosage compensation (DC). Three distinct molecular strategies to achieve DC have been previously described in nematodes, fruit flies, and mammals. Is this a consequence of distinct genomes, functional or ecological constraints, or random initial commitment to an evolutionary trajectory? Here, we study DC in the malaria mosquito Anopheles gambiae The Anopheles and Drosophila X chromosomes evolved independently but share a high degree of homology. We find that Anopheles achieves DC by a mechanism distinct from the Drosophila MSL complex-histone H4 lysine 16 acetylation pathway. CRISPR knockout of Anopheles msl-2 leads to embryonic lethality in both sexes. Transcriptome analyses indicate that this phenotype is not a consequence of defective X chromosome DC. By immunofluorescence and ChIP, H4K16ac does not preferentially enrich on the male X. Instead, the mosquito MSL pathway regulates conserved developmental genes. We conclude that a novel mechanism confers X chromosome up-regulation in Anopheles Our findings highlight the pluralism of gene-dosage buffering mechanisms even under similar genomic and functional constraints.

Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration.

Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5-/- HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.

Functional mechanisms and abnormalities of the nuclear lamina.

Alterations in nuclear shape are present in human diseases and ageing. A compromised nuclear lamina is molecularly interlinked to altered chromatin functions and genomic instability. Whether these alterations are a cause or a consequence of the pathological state are important questions in biology. Here, we summarize the roles of nuclear envelope components in chromatin organization, phase separation and transcriptional and epigenetic regulation. Examining these functions in healthy backgrounds will guide us towards a better understanding of pathological alterations.

RNA nucleation by MSL2 induces selective X chromosome compartmentalization.

Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2CTD is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.

Nephronophthisis gene products display RNA-binding properties and are recruited to stress granules.

Mutations of cilia-associated molecules cause multiple developmental defects that are collectively termed ciliopathies. However, several ciliary proteins, involved in gating access to the cilium, also assume localizations at other cellular sites including the nucleus, where they participate in DNA damage responses to maintain tissue integrity. Molecular insight into how these molecules execute such diverse functions remains limited. A mass spectrometry screen for ANKS6-interacting proteins suggested an involvement of ANKS6 in RNA processing and/or binding. Comparing the RNA-binding properties of the known RNA-binding protein BICC1 with the three ankyrin-repeat proteins ANKS3, ANKS6 (NPHP16) and INVERSIN (NPHP2) confirmed that certain nephronophthisis (NPH) family members can interact with RNA molecules. We also observed that BICC1 and INVERSIN associate with stress granules in response to translational inhibition. Furthermore, BICC1 recruits ANKS3 and ANKS6 into TIA-1-positive stress granules after exposure to hippuristanol. Our findings uncover a novel function of NPH family members, and provide further evidence that NPH family members together with BICC1 are involved in stress responses to maintain tissue and organ integrity.

Temporal expression of MOF acetyltransferase primes transcription factor networks for erythroid fate.

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are orchestrated by the combinatorial action of transcription factors and epigenetic regulators. Here, we have explored the mechanism by which histone H4 lysine 16 acetyltransferase MOF regulates erythropoiesis. Single-cell RNA sequencing and chromatin immunoprecipitation sequencing uncovered that MOF influences erythroid trajectory by dynamic recruitment to chromatin and its haploinsufficiency causes accumulation of a transient HSC population. A regulatory network consisting of MOF, RUNX1, and GFI1B is critical for erythroid fate commitment. GFI1B acts as a Mof activator which is necessary and sufficient for cell type-specific induction of Mof expression. Plasticity of Mof-depleted HSCs can be rescued by expression of a downstream effector, Gata1, or by rebalancing acetylation via a histone deacetylase inhibitor. Accurate timing and dosage of Mof expression act as a rheostat for the feedforward transcription factor network that safeguards progression along the erythroid fate.

Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.

Intergenerationally Maintained Histone H4 Lysine 16 Acetylation Is Instructive for Future Gene Activation.

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.

Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation.

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.