Accelerating 3D genomics data analysis with Microcket.

The three-dimensional (3D) organization of genome is fundamental to cell biology. To explore 3D genome, emerging high-throughput approaches have produced billions of sequencing reads, which is challenging and time-consuming to analyze. Here we present Microcket, a package for mapping and extracting interacting pairs from 3D genomics data, including Hi-C, Micro-C, and derivant protocols. Microcket utilizes a unique read-stitch strategy that takes advantage of the long read cycles in modern DNA sequencers; benchmark evaluations reveal that Microcket runs much faster than the current tools along with improved mapping efficiency, and thus shows high potential in accelerating and enhancing the biological investigations into 3D genome. Microcket is freely available at https://github.com/hellosunking/Microcket .

Single cell multiomic analysis reveals diabetes-associated ß-cell heterogeneity driven by HNF1A.

Broad heterogeneity in pancreatic ß-cell function and morphology has been widely reported. However, determining which components of this cellular heterogeneity serve a diabetes-relevant function remains challenging. Here, we integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets and identify Type II Diabetes (T2D)-associated ß-cell heterogeneity at both transcriptomic and epigenomic levels. We develop a computational method to explicitly dissect the intra-donor and inter-donor heterogeneity between single ß-cells, which reflect distinct mechanisms of T2D pathogenesis. Integrative transcriptomic and epigenomic analysis identifies HNF1A as a principal driver of intra-donor heterogeneity between ß-cells from the same donors; HNF1A expression is also reduced in ß-cells from T2D donors. Interestingly, HNF1A activity in single ß-cells is significantly associated with lower Na+ currents and we nominate a HNF1A target, FXYD2, as the primary mitigator. Our study demonstrates the value of investigating disease-associated single-cell heterogeneity and provides new insights into the pathogenesis of T2D.

Novel correlative analysis identifies multiple genomic variations impacting ASD with macrocephaly.

Autism spectrum disorders (ASD) display both phenotypic and genetic heterogeneity, impeding the understanding of ASD and development of effective means of diagnosis and potential treatments. Genes affected by genomic variations for ASD converge in dozens of gene ontologies (GOs), but the relationship between the variations at the GO level have not been well elucidated. In the current study, multiple types of genomic variations were mapped to GOs and correlations among GOs were measured in ASD and control samples. Several ASD-unique GO correlations were found, suggesting the importance of co-occurrence of genomic variations in genes from different functional categories in ASD etiology. Combined with experimental data, several variations related to WNT signaling, neuron development, synapse morphology/function and organ morphogenesis were found to be important for ASD with macrocephaly, and novel co-occurrence patterns of them in ASD patients were found. Furthermore, we applied this gene ontology correlation analysis method to find genomic variations that contribute to ASD etiology in combination with changes in gene expression and transcription factor binding, providing novel insights into ASD with macrocephaly and a new methodology for the analysis of genomic variation.

Easy Hi-C: A Low-Input Method for Capturing Genome Organization.

Chromosome conformation capture technology and its derivatives have been widely used to study genome organization. Among them, Hi-C (chromosome conformation capture coupling with high-throughput sequencing) is popular in dissecting chromatin architecture on the genome-wide level. However, the intrinsic limitations prevent its application when it comes to rare samples. Here, we present easy Hi-C, a biotin-free technology that dramatically reduces DNA loss and is suitable for low-input samples.

DeepLoop robustly maps chromatin interactions from sparse allele-resolved or single-cell Hi-C data at kilobase resolution.

Mapping chromatin loops from noisy Hi-C heatmaps remains a major challenge. Here we present DeepLoop, which performs rigorous bias correction followed by deep-learning-based signal enhancement for robust chromatin interaction mapping from low-depth Hi-C data. DeepLoop enables loop-resolution, single-cell Hi-C analysis. It also achieves a cross-platform convergence between different Hi-C protocols and micrococcal nuclease (micro-C). DeepLoop allowed us to map the genetic and epigenetic determinants of allele-specific chromatin interactions in the human genome. We nominate new loci with allele-specific interactions governed by imprinting or allelic DNA methylation. We also discovered that, in the inactivated X chromosome (Xi), local loops at the DXZ4 'megadomain' boundary escape X-inactivation but the FIRRE 'superloop' locus does not. Importantly, DeepLoop can pinpoint heterozygous single-nucleotide polymorphisms and large structure variants that cause allelic chromatin loops, many of which rewire enhancers with transcription consequences. Taken together, DeepLoop expands the use of Hi-C to provide loop-resolution insights into the genetics of the three-dimensional genome.

INFIMA leverages multi-omics model organism data to identify effector genes of human GWAS variants.

Genome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA's superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/ .

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Long noncoding RNA SAM promotes myoblast proliferation through stabilizing Sugt1 and facilitating kinetochore assembly.

The functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify SAM (Sugt1 asssociated muscle) lncRNA that is enriched in the proliferating myoblasts. Global deletion of SAM has no overt effect on mice but impairs adult muscle regeneration following acute damage; it also exacerbates the chronic injury-induced dystrophic phenotype in mdx mice. Consistently, inducible deletion of SAM in SCs leads to deficiency in muscle regeneration. Further examination reveals that SAM loss results in a cell-autonomous defect in the proliferative expansion of myoblasts. Mechanistically, we find SAM interacts and stabilizes Sugt1, a co-chaperon protein key to kinetochore assembly during cell division. Loss of SAM or Sugt1 both disrupts kinetochore assembly in mitotic cells due to the mislocalization of two components: Dsn1 and Hec1. Altogether, our findings identify SAM as a regulator of SC proliferation through facilitating Sugt1 mediated kinetochore assembly during cell division.

Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key ß-Cell-Specific Disease Genes.

Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct ß cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both ß-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity.

Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1ß/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.

Linc-YY1 promotes myogenic differentiation and muscle regeneration through an interaction with the transcription factor YY1.

Little is known how lincRNAs are involved in skeletal myogenesis. Here we describe the discovery of Linc-YY1 from the promoter of the transcription factor (TF) Yin Yang 1 (YY1) gene. We demonstrate that Linc-YY1 is dynamically regulated during myogenesis in vitro and in vivo. Gain or loss of function of Linc-YY1 in C2C12 myoblasts or muscle satellite cells alters myogenic differentiation and in injured muscles has an impact on the course of regeneration. Linc-YY1 interacts with YY1 through its middle domain, to evict YY1/Polycomb repressive complex (PRC2) from target promoters, thus activating the gene expression in trans. In addition, Linc-YY1 also regulates PRC2-independent function of YY1. Finally, we identify a human Linc-YY1 orthologue with conserved function and show that many human and mouse TF genes are associated with lincRNAs that may modulate their activity. Altogether, we show that Linc-YY1 regulates skeletal myogenesis and uncover a previously unappreciated mechanism of gene regulation by lincRNA.

Transcriptional pause release is a rate-limiting step for somatic cell reprogramming.

Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release.

Genome-wide profiling of YY1 binding sites during skeletal myogenesis.

Skeletal muscle differentiation is regulated by a network of transcription factors, epigenetic regulators and noncoding RNAs. We have recently performed ChIP-seq experiments to explore the genome-wide binding of transcription factor YY1 in skeletal muscle cells. Our results identified thousands of YY1 binding peaks, underscoring its multifaceted functions in muscle cells. In particular, we identified a very high proportion of YY1 binding peaks residing in the intergenic regions, which led to the discovery of some novel lincRNAs under YY1 regulation. Here we describe the details of the ChIP-seq experiments and data analysis procedures associated with the study published by Lu et al. in the EMBO Journal in 2013 [1].

Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis.

Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and non-coding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome-wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)-seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co-occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2-independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non-coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle-associated lincRNA (Yam-1) that is positively regulated by YY1. Yam-1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam-1 functions through in cis regulation of miR-715, which in turn targets Wnt7b. Our findings not only provide the first genome-wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNA Yam-1.

Zinc-finger protein 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA transcription in gastric cancer.

OBJECTIVE: Zinc-finger protein 545 (ZNF545) is a member of the family of Krüppel-associated box-containing zinc-finger proteins. The aim of this study was to clarify its biological function as a tumour suppressor in gastric cancer. DESIGN: The biological function of ZNF545 was determined by cell growth and apoptosis assays. The ZNF545 target signal pathway was identified by promoter luciferase assay, northern blot, run-on transcription assay, chromatin immunoprecipitation and coimmunoprecipitation assays. The clinical application of ZNF545 was assessed in primary gastric cancers. RESULTS: ZNF545 was silenced or reduced in 16 out of 18 gastric cancer cell lines by promoter hypermethylation. Restoration of ZNF545 expression in gastric cancer cell lines suppressed cell proliferation and induced apoptosis. These effects of ZNF545 were attributed to inhibition of ribosomal RNA (rRNA) transcription. Inhibition of rRNA transcription by ZNF545 was further revealed to be associated with direct ribosomal DNA (rDNA) promoter binding, recruitment of the corepressor, heterochromatin protein 1ß, and reduction of trimethylated histone H3 at the Lys4 residue at the rDNA locus. ZNF545 methylation was detected in 51.9% (41/79) of gastric cancer tissues, 27.0% (20/74) of adjacent non-tumour gastric tissues (p=0.001), but none of 20 normal controls. Multivariate analysis revealed that patients with ZNF545 methylation had a significant decrease in overall survival. Kaplan-Meier survival curves showed that ZNF545 methylation was significantly associated with shortened survival in patients with stage I-II gastric cancer. CONCLUSIONS: ZNF545 acts as a functional tumour suppressor in gastric cancer by inhibiting rRNA transcription. Its methylation at early stages of gastric carcinogenesis is an independent prognostic factor.

Pax3/7BP is a Pax7- and Pax3-binding protein that regulates the proliferation of muscle precursor cells by an epigenetic mechanism.

In mouse skeletal muscles, Pax7 uniquely marks muscle satellite cells and plays some important yet unknown functions at the perinatal stage. To elucidate its in vivo functions, we initiated a yeast two-hybrid screening to look for Pax7-interacting proteins and identified a previously uncharacterized Pax7- and Pax3-binding protein (Pax3/7BP). Pax3/7BP is a ubiquitously expressed nuclear protein, enriched in Pax7+ muscle precursor cells (MPCs), and serves as an indispensable adaptor for Pax7 to recruit the histone 3 lysine 4 (H3K4) methyltransferase (HMT) complex by bridging Pax7 and Wdr5. Knockdown of Pax3/7BP abolished the Pax3/7-associated H3K4 HMT activity and inhibited the proliferation of Pax7+ MPCs from young mice both in culture and in vivo. Id3 and Cdc20 were direct target genes of Pax7 and Pax3/7BP involved in the proliferation of Pax7+ MPCs. Collectively, our work establishes Pax3/7BP as an essential adaptor linking Pax3/7 with the H3K4 HMT to regulate the proliferation of MPCs.

A novel target of microRNA-29, Ring1 and YY1-binding protein (Rybp), negatively regulates skeletal myogenesis.

Skeletal muscle cell differentiation (myogenesis) is a process orchestrated by a complex network involving transcription factors, epigenetic regulators, and microRNAs. Previous studies identified miR-29 as a pro-myogenic factor that interacts with components of Polycomb repressive complex, YY1 and Ezh2. In a genome-wide survey of miR-29-mediated transcriptome changes in C2C12 myoblasts, many epigenetic factors were found to be down-regulated by miR-29. Among them, Rybp was shown to be a direct target of miR-29 through binding to its 3' UTR. Functional studies demonstrated that Rybp is down-regulated during myogenesis and acts as a negative regulator of skeletal myogenesis both in vitro during C2C12 differentiation and in vivo in injury-induced muscle regeneration. Furthermore, we found that Rybp and YY1 co-occupy several myogenic loci, including miR-29 itself, to silence their expression, thus forming a Rybp-miR-29 feedback loop. Rybp overexpression was found to enhance the enrichment of Ezh2 and trimethylation of H3K27 at target loci, suggesting it may facilitate the recruitment or stabilization of the Polycomb repressive complex. Collectively, our results identify Rybp as a novel regulator of myogenesis that co-acts with YY1 to silence miR-29 and other myogenic loci.

Inhibition of miR-29 by TGF-beta-Smad3 signaling through dual mechanisms promotes transdifferentiation of mouse myoblasts into myofibroblasts.

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in regulating many biological processes, including myogenic differentiation and muscle development. miR-29 is a promoting factor during myogenesis but its full spectrum of impact on muscle cells has yet to be explored. Here we describe an analysis of miR-29 affected transcriptome in C2C12 muscle cells using a high throughput RNA-sequencing platform. The results reveal that miR-29 not only functions to promote myogenic differentiation but also suppresses the transdifferentiation of myoblasts into myofibroblasts. miR-29 inhibits the fibrogenic differentiation through down-regulating both extracellular matrix genes and cell adhesion genes. We further demonstrate that miR-29 is under negative regulation by TGF-beta (TGF-ß)-Smad3 signaling via dual mechanisms of both inhibiting MyoD binding and enhancing Yin Yang 1 (YY1)-recruited Polycomb association. Together, these results identify miR-29 as a pleiotropic molecule in both myogenic and fibrogenic differentiation of muscle cells.

Loss of miR-29 in myoblasts contributes to dystrophic muscle pathogenesis.

microRNAs (miRNAs) are noncoding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in a multitude of physiological and pathological processes. Here, we describe the regulation and function of miR-29 in Duchenne muscular dystrophy (DMD) and its potential use as therapeutic target. Our results demonstrate that miR-29 expression is downregulated in dystrophic muscles of mdx mice, a model of DMD. Restoration of its expression by intramuscular and intravenous injection improved dystrophy pathology by both promoting regeneration and inhibiting fibrogenesis. Mechanistic studies revealed that loss of miR-29 in muscle precursor cells (myoblasts) promotes their transdifferentiation into myofibroblasts through targeting extracellular molecules including collagens and microfibrillar-associated protein 5 (Mfap5). We further demonstrated that miR-29 is under negative regulation by transforming growth factor-ß (TGF-ß) signaling. Together, these results not only identify TGF-ß-miR-29 as a novel regulatory axis during myoblasts conversion into myofibroblasts which constitutes a novel contributing route to muscle fibrogenesis of DMD but also implicate miR-29 replacement therapy as a promising treatment approach for DMD.