Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.

Activity-Induced MeCP2 Phosphorylation Regulates Retinogeniculate Synapse Refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENT: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.

HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes.

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.

Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing.

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.

Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation.

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.

Network Analysis Identifies Mitochondrial Regulation of Epidermal Differentiation by MPZL3 and FDXR.

Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.

CALML5 is a ZNF750- and TINCR-induced protein that binds stratifin to regulate epidermal differentiation.

Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750-TINCR-CALML5-SFN network is thus essential for epidermal differentiation.

A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation.

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.

ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼ 10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.

ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation.

Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.

SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.

Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair.

The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor.