TDP-43 chronic deficiency leads to dysregulation of transposable elements and gene expression by affecting R-loop and 5hmC crosstalk.

TDP-43 is an RNA/DNA-binding protein that forms aggregates in various brain disorders. TDP-43 engages in many aspects of RNA metabolism, but its molecular roles in regulating genes and transposable elements (TEs) have not been extensively explored. Chronic TDP-43 knockdown impairs cell proliferation and cellular responses to DNA damage. At the molecular level, TDP-43 chronic deficiency affects gene expression either locally or distally by concomitantly altering the crosstalk between R-loops and 5-hydroxymethylcytosine (5hmC) in gene bodies and long-range enhancer/promoter interactions. Furthermore, TDP-43 knockdown induces substantial disease-relevant TE activation by influencing their R-loop and 5hmC homeostasis in a locus-specific manner. Together, our findings highlight the genomic roles of TDP-43 in modulating R-loop-5hmC coordination in coding genes, distal regulatory elements, and TEs, presenting a general and broad molecular mechanism underlying the contributions of proteinopathies to the etiology of neurodegenerative disorders.

Ectopic transcription due to inappropriately inherited histone methylation may interfere with the ongoing function of terminally differentiated cells.

Many human neurodevelopmental disorders are caused by de novo mutations in histone modifying enzymes. These patients have craniofacial defects, developmental delay, intellectual disability and behavioral abnormalities, but it remains unclear how the mutations lead to such developmental defects. Here we take advantage of the invariant C. elegans lineage along with a unique double mutant in the H3K4me1/2 demethylase SPR-5/LSD1/KDM1A and the H3K9 methyltransferase MET-2/SETDB1 to address this question. We demonstrate that spr-5; met-2 double mutant worms have a severe chemotaxis defect that is dependent upon the ectopic expression of germline genes in somatic tissues. In addition, by performing single-cell RNAseq, we find that germline genes begin to be ectopically expression widely in spr-5; met-2 embryos. However, surprisingly we found that spr-5; met-2 mutants have no somatic lineage defects prior to the 200-cell stage of embryogenesis. This suggests that the altered chemotaxis behavior may be due to ongoing defect in terminally differentiated cells rather than a defect in development. To test this directly, we used RNAi to shut off the ectopic expression of germline genes in L2 spr-5; met-2 larvae, which have a fully formed nervous system. Remarkably, we find that shutting off the ectopic germline expression rescues normal chemotaxis behavior in the same adult worms that previously had a chemotaxis defect at the L2 stage. This suggests that ongoing ectopic transcription can block normal behavior in a fully intact nervous system. These data raise the possibility that intellectual disability and altered behavior in neurodevelopmental syndromes, caused by mutations in histone modifying enzymes, could be due to ongoing ectopic transcription and may be reversible.

Regulation of CTCF loop formation during pancreatic cell differentiation.

Transcription reprogramming during cell differentiation involves targeting enhancers to genes responsible for establishment of cell fates. To understand the contribution of CTCF-mediated chromatin organization to cell lineage commitment, we analyzed 3D chromatin architecture during the differentiation of human embryonic stem cells into pancreatic islet organoids. We find that CTCF loops are formed and disassembled at different stages of the differentiation process by either recruitment of CTCF to new anchor sites or use of pre-existing sites not previously involved in loop formation. Recruitment of CTCF to new sites in the genome involves demethylation of H3K9me3 to H3K9me2, demethylation of DNA, recruitment of pioneer factors, and positioning of nucleosomes flanking the new CTCF sites. Existing CTCF sites not involved in loop formation become functional loop anchors via the establishment of new cohesin loading sites containing NIPBL and YY1 at sites between the new anchors. In both cases, formation of new CTCF loops leads to strengthening of enhancer promoter interactions and increased transcription of genes adjacent to loop anchors. These results suggest an important role for CTCF and cohesin in controlling gene expression during cell differentiation.

Characterization of a strain-specific CD-1 reference genome reveals potential inter- and intra-strain functional variability.

BACKGROUND: CD-1 is an outbred mouse stock that is frequently used in toxicology, pharmacology, and fundamental biomedical research. Although inbred strains are typically better suited for such studies due to minimal genetic variability, outbred stocks confer practical advantages over inbred strains, such as improved breeding performance and low cost. Knowledge of the full genetic variability of CD-1 would make it more useful in toxicology, pharmacology, and fundamental biomedical research. RESULTS: We performed deep genomic DNA sequencing of CD-1 mice and used the data to identify genome-wide SNPs, indels, and germline transposable elements relative to the mm10 reference genome. We used multiple genome-wide sequencing data types and previously published CD-1 SNPs to validate our called variants. We used the called variants to construct a strain-specific CD-1 reference genome, which we show can improve mappability and reduce experimental biases from genome-wide sequencing data derived from CD-1 mice. Based on previously published ChIP-seq and ATAC-seq data, we find evidence that genetic variation between CD-1 mice can lead to alterations in transcription factor binding. We also identified a number of variants in the coding region of genes which could have effects on translation of genes. CONCLUSIONS: We have identified millions of previously unidentified CD-1 variants with the potential to confound studies involving CD-1. We used the identified variants to construct a CD-1-specific reference genome, which can improve accuracy and reduce bias when aligning genomics data derived from CD-1 mice.

Structures of CTCF-DNA complexes including all 11 zinc fingers.

The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1-ZF7 and ZF3-ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1-ZF7 follow the right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three base pairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9-ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1-ZF7 and ZF8-ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1-ZF7 and bulkier and polar/charged residues for ZF8-ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine-guanine and glutamine-adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9-ZF11. The differences between ZF1-ZF7 and ZF8-ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

Single nucleus multiome analysis of the prefrontal cortex from C9orf72 ALS/FTD patients illuminates pathways affected during disease progression.

Repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and familial frontotemporal dementia (ALS/FTD). To identify molecular defects that take place in the dorsolateral frontal cortex of patients with C9orf72 ALS/FTD, we compared healthy controls with C9orf72 ALS/FTD donor samples staged based on the levels of cortical phosphorylated TAR DNA binding protein (pTDP-43), a neuropathological hallmark of disease progression. We identified distinct molecular changes in different cell types that take place during disease progression. These alterations include downregulation of nuclear and mitochondrial ribosomal protein genes in early disease stages that become upregulated as the disease progresses. High ratios of premature oligodendrocytes expressing low levels of genes encoding major myelin protein components are characteristic of late disease stages and may represent a unique signature of C9orf72 ALS/FTD. Microglia with increased reactivity and astrocyte specific transcriptome changes in genes involved in glucose/glycogen metabolism are also associated with disease progression. Late stages of C9orf72 ALS/FTD correlate with sequential changes in the regulatory landscape of several genes in glial cells, namely MBP/MAG/MOG in oligodendrocytes, CD83/IRF8 in microglia, and GLUT1/GYS2/AGL in astrocytes. Only layer 2-3 cortical projection neurons with high expression of CUX2/LAMP5 are significantly reduced in C9orf72 ALS/FTD patients with respect to controls. Our findings reveal previously unknown progressive functional changes in cortical cells of C9orf72 ALS/FTD patients that shed light on the mechanisms underlying the pathology of this disease.

Expansion of the genotypic and phenotypic spectrum of CTCF-related disorder guides clinical management: 43 new subjects and a comprehensive literature review.

Monoallelic variants of CTCF cause an autosomal dominant neurodevelopmental disorder with a wide range of features, including impacts on the brain, growth, and craniofacial development. A growing number of subjects with CTCF-related disorder (CRD) have been identified due to the increased application of exome sequencing, and further delineation of the clinical spectrum of CRD is needed. Here, we examined the clinical features, including facial profiles, and genotypic spectrum of 107 subjects with identified CTCF variants, including 43 new and 64 previously described subjects. Among the 43 new subjects, 23 novel variants were reported. The cardinal clinical features in subjects with CRD included intellectual disability/developmental delay (91%) with speech delay (65%), motor delay (53%), feeding difficulties/failure to thrive (66%), ocular abnormalities (56%), musculoskeletal anomalies (53%), and behavioral problems (52%). Other congenital anomalies were also reported, but none of them were common. Our findings expanded the genotypic and phenotypic spectrum of CRD that will guide genetic counseling, management, and surveillance care for patients with CRD. Additionally, a newly built facial gestalt on the Face2Gene tool will facilitate prompt recognition of CRD by physicians and shorten a patient's diagnostic odyssey.

Recruitment of CTCF to an Fto enhancer is responsible for transgenerational inheritance of BPA-induced obesity.

The mechanisms by which environmentally-induced epiphenotypes are transmitted transgenerationally in mammals are poorly understood. Here we show that exposure of pregnant mouse females to bisphenol A (BPA) results in obesity in the F2 progeny due to increased food intake. This epiphenotype can be transmitted up to the F6 generation. Analysis of chromatin accessibility in sperm of the F1-F6 generations reveals alterations at sites containing binding motifs for CCCTC-binding factor (CTCF) at two cis-regulatory elements (CREs) of the Fto gene that correlate with transmission of obesity. These CREs show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite-controlling neurons. Deletion of the CTCF site in Fto results in mice that have normal food intake and fail to become obese when ancestrally exposed to BPA. The results suggest that epigenetic alterations of Fto can lead to the same phenotypes as genetic variants.

Activation of HIV-1 proviruses increases downstream chromatin accessibility.

It is unclear how the activation of HIV-1 transcription affects chromatin structure. We interrogated chromatin organization both genome-wide and nearby HIV-1 integration sites using Hi-C and ATAC-seq. In conjunction, we analyzed the transcription of the HIV-1 genome and neighboring genes. We found that long-range chromatin contacts did not differ significantly between uninfected cells and those harboring an integrated HIV-1 genome, whether the HIV-1 genome was actively transcribed or inactive. Instead, the activation of HIV-1 transcription changes chromatin accessibility immediately downstream of the provirus, demonstrating that HIV-1 can alter local cellular chromatin structure. Finally, we examined HIV-1 and neighboring host gene transcripts with long-read sequencing and found populations of chimeric RNAs both virus-to-host and host-to-virus. Thus, multiomics profiling revealed that the activation of HIV-1 transcription led to local changes in chromatin organization and altered the expression of neighboring host genes.

The Cupid shuffle: Do enhancers prefer specific promoters?

Two recent reports (Martinez-Ara et al., 2022; Bergman et al., 2022) explore the compatibility between enhancers and promoters and find that enhancers preferentially activate promoters with low intrinsic activity rather than favoring housekeeping or cell-type-specific promoters.

Indolium 1 Exerts Activity against Vemurafenib-Resistant Melanoma In Vivo.

The development of targeted therapies (BRAF/MEK inhibitors) and immunotherapy have had a major impact on the treatment of melanoma. However, the majority of patients with advanced melanomas succumb to their disease. The mechanisms of resistance to both targeted therapies and immunotherapies are numerous and have been well-described. These include the alternative activation of BRAF/MEK signaling, novel compensating mutations in additional oncogenes, and loss of neoantigens. There has been limited development of small molecules that target alternative pathways in melanoma in the last two decades. We have previously identified triphenylmethanes as a class that shows activity against a wide variety of tumors. We have synthesized a novel triphenylmethane, indolium 1, and demonstrated its efficacy against an aggressive vemurafenib-resistant melanoma in vivo. Indolium 1 has a novel mechanism of action against melanoma, in that it results in induction of the tumor-suppressor EPHA3. We believe that pre-IND studies are warranted for this novel compound, given its mechanism of action and ability to inhibit the growth of vemurafenib resistant melanoma in vivo.

Introduction of Mutant GNAQ into Endothelial Cells Induces a Vascular Malformation Phenotype with Therapeutic Response to Imatinib.

GNAQ is mutated in vascular and melanocytic lesions, including vascular malformations and nevi. No in vivo model of GNAQ activation in endothelial cells has previously been described. We introduce mutant GNAQ into a murine endothelial cell line, MS1. The resultant transduced cells exhibit a novel phenotype in vivo, with extensive vasoformative endothelial cells forming aberrant lumens similar to those seen in vascular malformations. ATAC-seq analysis reveals activation of c-Kit in the novel vascular malformations. We demonstrate that c-Kit is expressed in authentic human Sturge-Weber vascular malformations, indicating a novel druggable target for Sturge-Weber syndrome. Since c-Kit is targeted by the FDA-approved drug imatinib, we tested the ability of imatinib on the phenotype of the vascular malformations in vivo. Imatinib treated vascular malformations are significantly smaller and have decreased supporting stromal cells surrounding the lumen. Imatinib may be useful in the treatment of human vascular malformations that express c-Kit, including Sturge-Weber syndrome.

Beyond Genes: Germline Disruption in the Etiology of Autism Spectrum Disorders.

Investigations into the etiology of autism spectrum disorders have been largely confined to two realms: variations in DNA sequence and somatic developmental exposures. Here we suggest a third route-disruption of the germline epigenome induced by exogenous toxicants during a parent's gamete development. Similar to cases of germline mutation, these molecular perturbations may produce dysregulated transcription of brain-related genes during fetal and early development, resulting in abnormal neurobehavioral phenotypes in offspring. Many types of exposures may have these impacts, and here we discuss examples of anesthetic gases, tobacco components, synthetic steroids, and valproic acid. Alterations in parental germline could help explain some unsolved phenomena of autism, including increased prevalence, missing heritability, skewed sex ratio, and heterogeneity of neurobiology and behavior.

Is developmental synchrony enabled by CTCF residence time?

Depletion of CTCF in cultured cells has minor effects on transcription whereas its mutation leads to embryonic lethality and developmental defects. In a recent issue of Nature Cell Biology, Soochit et al. (2021) show that the residence time of CTCF on DNA may explain its critical role in cell differentiation.

Principles of 3D compartmentalization of the human genome.

Chromatin is organized in the nucleus via CTCF loops and compartmental domains. Here, we compare different cell types to identify distinct paradigms of compartmental domain formation in human tissues. We identify and quantify compartmental forces correlated with histone modifications characteristic of transcriptional activity and previously underappreciated roles for distinct compartmental domains correlated with the presence of H3K27me3 and H3K9me3, respectively. We present a computer simulation model capable of predicting compartmental organization based on the biochemical characteristics of independent chromatin features. Using this model, we show that the underlying forces responsible for compartmental domain formation in human cells are conserved and that the diverse compartmentalization patterns seen across cell types are due to differences in chromatin features. We extend these findings to Drosophila to suggest that the same principles are at work beyond humans. These results offer mechanistic insights into the fundamental forces driving the 3D organization of the genome.

Exposure to sevoflurane results in changes of transcription factor occupancy in sperm and inheritance of autism†.

One in 54 children in the United States is diagnosed with autism spectrum disorder. De novo germline and somatic mutations cannot account for all cases of autism spectrum disorder, suggesting that epigenetic alterations triggered by environmental exposures may be responsible for a subset of autism spectrum disorder cases. Human and animal studies have shown that exposure of the developing brain to general anesthetic agents can trigger neurodegeneration and neurobehavioral abnormalities, but the effects of general anesthetics on the germline have not been explored in detail. We exposed pregnant mice to sevoflurane during the time of embryonic development when the germ cells undergo epigenetic reprogramming and found that more than 38% of the directly exposed F1 animals exhibit impairments in anxiety and social interactions. Strikingly, 44-47% of the F2 and F3 animals, which were not directly exposed to sevoflurane, show the same behavioral problems. We performed ATAC-seq and identified more than 1200 differentially accessible sites in the sperm of F1 animals, 69 of which are also present in the sperm of F2 animals. These sites are located in regulatory regions of genes strongly associated with autism spectrum disorder, including Arid1b, Ntrk2, and Stmn2. These findings suggest that epimutations caused by exposing germ cells to sevoflurane can lead to autism spectrum disorder in the offspring, and this effect can be transmitted through the male germline inter- and transgenerationally.

Replication timing maintains the global epigenetic state in human cells.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

Regulation of 3D chromatin organization by CTCF.

Studies of nuclear architecture using chromosome conformation capture methods have provided a detailed view of how chromatin folds in the 3D nuclear space. New variants of this technology now afford unprecedented resolution and allow the identification of ever smaller folding domains that offer new insights into the mechanisms by which this organization is established and maintained. Here we review recent results in this rapidly evolving field with an emphasis on CTCF function, with the goal of gaining a mechanistic understanding of the principles by which chromatin is folded in the eukaryotic nucleus.

Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing.

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.