RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

Characterization of the TBR1 interactome: variants associated with neurodevelopmental disorders disrupt novel protein interactions.

TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.

Compartmentalization of androgen receptors at endogenous genes in living cells.

A wide range of nuclear proteins are involved in the spatio-temporal organization of the genome through diverse biological processes such as gene transcription and DNA replication. Upon stimulation by testosterone and translocation to the nucleus, multiple androgen receptors (ARs) accumulate in microscopically discernable foci which are irregularly distributed in the nucleus. Here, we investigated the formation and physical nature of these foci, by combining novel fluorescent labeling techniques to visualize a defined chromatin locus of AR-regulated genes-PTPRN2 or BANP-simultaneously with either AR foci or individual AR molecules. Quantitative colocalization analysis showed evidence of AR foci formation induced by R1881 at both PTPRN2 and BANP loci. Furthermore, single-particle tracking (SPT) revealed three distinct subdiffusive fractional Brownian motion (fBm) states: immobilized ARs were observed near the labeled genes likely as a consequence of DNA-binding, while the intermediate confined state showed a similar spatial behavior but with larger displacements, suggesting compartmentalization by liquid-liquid phase separation (LLPS), while freely mobile ARs were diffusing in the nuclear environment. All together, we show for the first time in living cells the presence of AR-regulated genes in AR foci.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Proteomic analysis of FOXP proteins reveals interactions between cortical transcription factors associated with neurodevelopmental disorders.

FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes.

Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.

The pluripotent genome in three dimensions is shaped around pluripotency factors.

It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal.

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog-Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog-Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2-Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal.

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

RTTN mutations link primary cilia function to organization of the human cerebral cortex.

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells.

Polycomb group (PcG) proteins play an important role in the control of developmental gene expression in higher organisms. In mammalian systems, PcG proteins participate in the control of pluripotency, cell fate, cell cycle regulation, X chromosome inactivation and parental imprinting. In this study we have analysed the function of the mouse PcG protein polycomblike 2 (Pcl2), one of three homologues of the Drosophila Polycomblike (Pcl) protein. We show that Pcl2 is expressed at high levels during early embryogenesis and in embryonic stem (ES) cells. At the biochemical level, Pcl2 interacts with core components of the histone H3K27 methyltransferase complex Polycomb repressive complex 2 (PRC2), to form a distinct substoichiometric biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 facilitates both PRC2 recruitment to the inactive X chromosome in differentiating XX ES cells and PRC2 recruitment to target genes in undifferentiated ES cells. The role of Pcl2 in PRC2 targeting in ES cells is critically dependent on a conserved PHD finger domain, suggesting that Pcl2 might function through the recognition of a specific chromatin configuration.

Genetic variants for head size share genes and pathways with cancer.

The size of the human head is highly heritable, but genetic drivers of its variation within the general population remain unmapped. We perform a genome-wide association study on head size (N = 80,890) and identify 67 genetic loci, of which 50 are novel. Neuroimaging studies show that 17 variants affect specific brain areas, but most have widespread effects. Gene set enrichment is observed for various cancers and the p53, Wnt, and ErbB signaling pathways. Genes harboring lead variants are enriched for macrocephaly syndrome genes (37-fold) and high-fidelity cancer genes (9-fold), which is not seen for human height variants. Head size variants are also near genes preferentially expressed in intermediate progenitor cells, neural cells linked to evolutionary brain expansion. Our results indicate that genes regulating early brain and cranial growth incline to neoplasia later in life, irrespective of height. This warrants investigation of clinical implications of the link between head size and cancer.

Disease modeling using embryonic stem cells: MeCP2 regulates nuclear size and RNA synthesis in neurons.

Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2.

An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin.

The mechanism by which the eukaryotic DNA-replication machinery penetrates condensed chromatin structures to replicate the underlying DNA is poorly understood. Here we provide evidence that an ACF1-ISWI chromatin-remodeling complex is required for replication through heterochromatin in mammalian cells. ACF1 (ATP-utilizing chromatin assembly and remodeling factor 1) and an ISWI isoform, SNF2H (sucrose nonfermenting-2 homolog), become specifically enriched in replicating pericentromeric heterochromatin. RNAi-mediated depletion of ACF1 specifically impairs the replication of pericentromeric heterochromatin. Accordingly, depletion of ACF1 causes a delay in cell-cycle progression through the late stages of S phase. In vivo depletion of SNF2H slows the progression of DNA replication throughout S phase, indicating a functional overlap with ACF1. Decondensing the heterochromatin with 5-aza-2-deoxycytidine reverses the effects of ACF1 and SNF2H depletion. Expression of an ACF1 mutant that cannot interact with SNF2H also interferes with replication of condensed chromatin. Our data suggest that an ACF1-SNF2H complex is part of a dedicated mechanism that enables DNA replication through highly condensed regions of chromatin.

Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia is a structural birth defect of the diaphragm, with lung hypoplasia and persistent pulmonary hypertension. Aside from vascular defects, the lungs show a disturbed balance of differentiated airway epithelial cells. The Sry related HMG box protein SOX2 is an important transcription factor for proper differentiation of the lung epithelium. The transcriptional activity of SOX2 depends on interaction with other proteins and the identification of SOX2-associating factors may reveal important complexes involved in the disturbed differentiation in CDH. To identify SOX2-associating proteins, we purified SOX2 complexes from embryonic mouse lungs at 18.5 days of gestation. Mass spectrometry analysis of SOX2-associated proteins identified several potential candidates, among which were the Chromodomain Helicase DNA binding protein 4 (CHD4), Cut-Like Homeobox1 (CUX1), and the Forkhead box proteins FOXP2 and FOXP4. We analyzed the expression patterns of FOXP2, FOXP4, CHD4, and CUX1 in lung during development and showed co-localization with SOX2. Co-immunoprecipitations validated the interactions of these four transcription factors with SOX2, and large-scale chromatin immunoprecipitation (ChIP) data indicated that SOX2 and CHD4 bound to unique sites in the genome, but also co-occupied identical regions, suggesting that these complexes could be involved in co-regulation of genes involved in the respiratory system.

The Williams syndrome transcription factor interacts with PCNA to target chromatin remodelling by ISWI to replication foci.

Chromatin states have to be faithfully duplicated during DNA replication to maintain cell identity. It is unclear whether or how ATP-dependent chromatin-remodelling factors are involved in this process. Here we provide evidence that the Williams syndrome transcription factor (WSTF) is targeted to replication foci through direct interaction with the DNA clamp PCNA, an important coordinator of DNA and chromatin replication. WSTF, in turn, recruits imitation switch (ISWI)-type nucleosome-remodelling factor SNF2H to replication sites. These findings reveal a novel recruitment mechanism for ATP-dependent chromatin-remodelling factors that is fundamentally different from the previously documented targeting by sequence-specific transcriptional regulators. RNA-interference-mediated depletion of WSTF or SNF2H causes a compaction of newly replicated chromatin and increases the amount of heterochromatin markers, including HP1beta. This increase in the amount of HP1beta protein is mediated by progression through S phase and is not the result of an increase in HP1beta mRNA levels. We propose that the WSTF-ISWI complex has a role in the maintenance of chromatin structures during DNA replication.

Publisher Correction: Mediator complex interaction partners organize the transcriptional network that defines neural stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.