Multi-omics analysis for potential inflammation-related genes involved in tumor immune evasion via extended application of epigenetic data

Background: Accumulating evidence suggests that inflammation-related genes may play key roles in tumor immune evasion. Programmed cell death ligand 1 (PD-L1) is an important immune checkpoint involved in mediating antitumor immunity. We performed multi-omics analysis to explore key inflammation-related genes affecting the transcriptional regulation of PD-L1 expression. Methods: The open chromatin region of the PD-L1 promoter was mapped using the assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles. Correlation analysis of epigenetic data (ATAC-seq) and transcriptome data (RNA-seq) were performed to identify inflammation-related transcription factors whose expression levels were correlated with the chromatin accessibility of the PD-L1 promoter. Chromatin immunoprecipitation sequencing (ChIP-seq) profiles were used to confirm the physical binding of the TF STAT2 and the predicted binding regions. We also confirmed the results of the bioinformatics analysis with cell experiments. Results: We identified chr9:5449463-5449962 and chr9:5450250-5450749 as reproducible open chromatin regions in the PD-L1 promoter. Moreover, we observed a correlation between STAT2 expression and the accessibility of the aforementioned regions. Furthermore, we confirmed its physical binding through ChIP-seq profiles and demonstrated the regulation of PD-L1 by STAT2 overexpression in vitro. Multiple databases were also used for the validation of the results. Conclusion: Our study identified STAT2 as a direct upstream TF regulating PD-L1 expression. The interaction of STAT2 and PD-L1 might be associated with tumor immune evasion in cancers, suggesting the potential value for tumor treatment.

SETD2-mediated epigenetic regulation of noncanonical Wnt5A during osteoclastogenesis

To define the role of SETD2 in the WNT5a signaling in the context of osteoclastogenesis, we exploited two different models: in vitro osteoclast differentiation, and K/BxN serum-induced arthritis model. We found that SETD2 and WNT5a were upregulated during osteoclast differentiation and after induction of arthritis. Using gain- and loss-of-function approaches in the myeloid cell, we confirmed that SETD2 regulated the osteoclast markers, and WNT5a via modulating active histone marks by enriching H3K36me3, and by reducing repressive H3K27me3 mark. Additionally, during osteoclastic differentiation, the transcription of Wnt5a was also associated with the active histone H3K9 and H4K8 acetylations. Mechanistically, SETD2 directed induction of NF-κβ expression facilitated the recruitment of H3K9Ac and H4K8Ac around the TSS region of the Wnt5a gene, thereby, assisting osteoclast differentiation. Together these findings for the first time revealed that SETD2 mediated epigenetic regulation of Wnt5a plays a critical role in osteoclastogenesis and induced arthritis. Graphic abstract Model for the Role of SETD2 dependent regulation of osteoclastic differentiation. A In monocyte cells SETD2-dependent H3K36 trimethylation help to create open chromatin region along with active enhancer mark, H3K27Ac. This chromatin state facilitated the loss of a suppressive H3K27me3 mark. B Additionally, SETD2 mediated induction of NF-κβ expression leads to the recruitment of histone acetyl transferases, P300/PCAF, to the Wnt5a gene and establish H3K9Ac and H4K8Ac marks. Along with other activation marks, these acetylation marks help in Wnt5a transcription which leads to osteoclastogenesis.

Unbiased identification of novel transcription factors in striatal compartmentation and striosome maturation

Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1-EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2, and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.

Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring

The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.

CharPlant: A De Novo Open Chromatin Region (OCR) Prediction Tool for Plant Genomes

Chromatin accessibility is a highly informative structural feature for understanding gene transcription regulation because it indicates the degree to which nuclear macromolecules such as proteins and RNA can access chromosomal DNA. Studies show that chromatin accessibility is highly dynamic during stress response, stimulus response, and developmental transition. Moreover, physical access to chromosomal DNA in eukaryotes is highly cell-specific. Therefore, current technologies such as DNase-seq, ATAC-seq, and FAIRE-seq reveal only a portion of the open chromatin regions (OCRs) present in a given species. Thus, the genome-wide distribution of OCRs remains unknown. In this study, we developed a bioinformatics tool called CharPlant for the de novo prediction of chromatin accessible regions in plant genomes. To develop this tool, we constructed a three-layer convolutional neural network (CNN) and subsequently trained the CNN using DNase-seq and ATAC-seq datasets of four plant species. The model simultaneously learns the sequence motifs and regulatory logics, which are jointly used to determine DNA accessibility. All of these steps are integrated into CharPlant, which can be run using a simple command line. The results of data analysis using CharPlant in this study demonstrate its prediction power and computational efficiency. To our knowledge, CharPlant is the first de novo prediction tool that can identify potential OCRs in the whole genome. The source code of CharPlant and supporting files are freely downloadable from https://github.com/Yin-Shen/CharPlant.

Transcriptional and epigenetic characterization of early striosomes identifies Foxf2 and Olig2 as factors required for development of striatal compartmentation and neuronal phenotypic differentiation

The basal ganglia, best known for processing information required for multiple aspects of movement, is also part of a network which regulates reward and cognition. The major output nucleus of the basal ganglia is the striatum, and its functions are dependent on neuronal compartmentation, including striosomes and matrix, which are selectively affected in disease. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), all of which share basic molecular signatures but are subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating terminal neuronal differentiation following migration are largely unidentified. We performed RNA- and ATAC-seq on sorted murine striosome and matrix cells at postnatal day 3. Focusing on the striosomal compartment, we validated the localization and role of transcription factors and their regulator(s), previously not known to be associated with striatal development, including Irx1, Foxf2, Olig2 and Stat1/2. In addition, we validated the enhancer function of a striosome-specific open chromatin region located 15Kb downstream of the Olig2 gene. These data and data bases provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation and thus provide new approaches to establishing MSN subtypes from human iPSCs for disease modeling and drug discovery.

Architecture of promoters of house-keeping genes in polytene chromosome interbands of drosophila melanogaster

This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and “head-to-head” arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.

Epigenetic profiling of growth plate chondrocytes sheds insight into regulatory genetic variation influencing height

GWAS have identified hundreds of height-associated loci. However, determining causal mechanisms is challenging, especially since height-relevant tissues (e.g. growth plates) are difficult to study. To uncover mechanisms by which height GWAS variants function, we performed epigenetic profiling of murine femoral growth plates. The profiled open chromatin regions recapitulate known chondrocyte and skeletal biology, are enriched at height GWAS loci, particularly near differentially expressed growth plate genes, and enriched for binding motifs of transcription factors with roles in chondrocyte biology. At specific loci, our analyses identified compelling mechanisms for GWAS variants. For example, at CHSY1, we identified a candidate causal variant (rs9920291) overlapping an open chromatin region. Reporter assays demonstrated that rs9920291 shows allelic regulatory activity, and CRISPR/Cas9 targeting of human chondrocytes demonstrates that the region regulates CHSY1 expression. Thus, integrating biologically relevant epigenetic information (here, from growth plates) with genetic association results can identify biological mechanisms important for human growth.