Chromatin accessibility analysis and architectural profiling of human kidneys reveal key cell types and a regulator of diabetic kidney disease.

Diabetes is the leading cause of kidney disease that progresses to kidney failure. However, the key molecular and cellular pathways involved in diabetic kidney disease (DKD) pathogenesis are largely unknown. Here, we performed a comparative analysis of adult human kidneys by examining cell type-specific chromatin accessibility by single-nucleus ATAC-seq (snATAC-seq) and analyzing three-dimensional chromatin architecture via high-throughput chromosome conformation capture (Hi-C method) of paired samples. We mapped the cell type-specific and DKD-specific open chromatin landscape and found that genetic variants associated with kidney diseases were significantly enriched in the proximal tubule- (PT) and injured PT-specific open chromatin regions in samples from patients with DKD. BACH1 was identified as a core transcription factor of injured PT cells; its binding target genes were highly associated with fibrosis and inflammation, which were also key features of injured PT cells. Additionally, Hi-C analysis revealed global chromatin architectural changes in DKD, accompanied by changes in local open chromatin patterns. Combining the snATAC-seq and Hi-C data identified direct target genes of BACH1, and indicated that BACH1 binding regions showed increased chromatin contact frequency with promoters of their target genes in DKD. Thus, our multi-omics analysis revealed BACH1 target genes in injured PTs and highlighted the role of BACH1 as a novel regulator of tubular inflammation and fibrosis.

Identification of Cell Type-Specific Effects of DNMT3A Mutations on Relapse in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by distinctive mutations in individual patients; therefore, each patient may display different cell-type compositions. Although most patients with AML achieve complete remission (CR) through intensive chemotherapy, the likelihood of relapse remains high. Several studies have attempted to characterize the genetic and cellular heterogeneity of AML; however, our understanding of the cellular heterogeneity of AML remains limited. In this study, we performed single-cell RNA sequencing (scRNAseq) of bone marrow-derived mononuclear cells obtained from same patients at different AML stages (diagnosis, CR, and relapse). We found that hematopoietic stem cells (HSCs) at diagnosis were abnormal compared to normal HSCs. By improving the detection of the DNMT3A R882 mutation with targeted scRNAseq, we identified that DNMT3A-mutant cells that mainly remained were granulocyte-monocyte progenitors (GMPs) or lymphoid-primed multipotential progenitors (LMPPs) from CR to relapse and that DNMT3A-mutant cells have gene signatures related to AML and leukemic cells. Copy number variation analysis at the single-cell level indicated that the cell type that possesses DNMT3A mutations is an important factor in AML relapse and that GMP and LMPP cells can affect relapse in patients with AML. This study advances our understanding of the role of DNMT3A in AML relapse and our approach can be applied to predict treatment outcomes.

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

BACKGROUND: The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming. RESULTS: The comparative dissection of the transcription profiles and histone modification patterns at lysines 4 and 27 of histone H3 of the iPSC, iPSCp, ESC, and somatic cells revealed that the iPSC was almost completely comparable to the ESC, regardless of their origins, whereas the genes of the iPSCp were dysregulated to a larger extent. Regardless of the origins of the somatic cells, the fibroblasts induced using the ESC protein extracts appear to be completely reprogrammed into pluripotent cells, although they show unshared marginal differences in their gene expression programs, which may not affect the maintenance of stemness. A comparative investigation of the iPSCp generated by unwanted reprogramming showed that the two groups of genes on the pathway from somatic cells to iPSC might function as sequential reprogramming-competent early and late responders to the induction stimulus. Moreover, some of the divergent genes expressed only in the iPSCp were associated with many tumor-related pathways. CONCLUSIONS: Faithful transcriptional reprogramming should follow epigenetic alterations to generate induced pluripotent stem cells from somatic cells. This genome-wide comparison enabled us to define the early and late responder genes during the cell reprogramming process to iPSC. Our results indicate that the cellular responsiveness to external stimuli should be pre-determined and sequentially orchestrated through the tight modulation of the chromatin environment during cell reprogramming to prevent unexpected reprogramming.

Z-DNA-forming sites identified by ChIP-Seq are associated with actively transcribed regions in the human genome.

Z-DNA, a left-handed double helical DNA is structurally different from the most abundant B-DNA. Z-DNA has been known to play a significant role in transcription and genome stability but the biological meaning and positions of Z-DNA-forming sites (ZFSs) in the human genome has not been fully explored. To obtain genome-wide map of ZFSs, Zaa with two Z-DNA-binding domains was used for ChIP-Seq analysis. A total of 391 ZFSs were found and their functions were examined in vivo. A large portion of ZFSs was enriched in the promoter regions and contain sequences with high potential to form Z-DNA. Genes containing ZFSs were occupied by RNA polymerase II at the promoters and showed high levels of expression. Moreover, ZFSs were significantly related to active histone marks such as H3K4me3 and H3K9ac. The association of Z-DNA with active transcription was confirmed by the reporter assay system. Overall, our results suggest that Z-DNA formation depends on chromatin structure as well as sequence composition, and is associated with active transcription in human cells. The global information about ZFSs positioning will provide a useful resource for further understanding of DNA structure-dependent transcriptional regulation.

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes.

An extension of directed evolution strategies to genome-wide variations increases the chance of obtaining metabolite-overproducing microbes. However, a general high-throughput screening platform for selecting improved strains remains out of reach. Here, to expedite the evolution of metabolite-producing microbes, we utilize synthetic RNA devices comprising a riboswitch and a selection module that specifically sense inconspicuous metabolites. Using L-lysine-producing Escherichia coli as a model system, we demonstrated that this RNA device could enrich pathway-optimized strains to up to 75% of the total population after four rounds of enrichment cycles. Furthermore, the potential applicability of this device was examined by successfully extending its application to the case of L-tryptophan. When used in conjunction with combinatorial mutagenesis for metabolite overproduction, our synthetic RNA device should facilitate strain improvement.