Droplet-based single-cell joint profiling of histone modifications and transcriptomes.

We previously reported Paired-Tag, a combinatorial indexing-based method that can simultaneously map histone modifications and gene expression at single-cell resolution at scale. However, the lengthy procedure of Paired-Tag has hindered its general adoption in the community. To address this bottleneck, we developed a droplet-based Paired-Tag protocol that is faster and more accessible than the previous method. Using cultured mammalian cells and primary brain tissues, we demonstrate its superior performance at identifying candidate cis-regulatory elements and associating their dynamic chromatin state to target gene expression in each constituent cell type in a complex tissue.

The microbiota regulates hematopoietic stem and progenitor cell development by mediating inflammatory signals in the niche.

The commensal microbiota regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. Whether and how the microbiota influences HSPC development during embryogenesis is unclear. Using gnotobiotic zebrafish, we show that the microbiota is necessary for HSPC development and differentiation. Individual bacterial strains differentially affect HSPC formation, independent of their effects on myeloid cells. Early-life dysbiosis in chd8-/- zebrafish impairs HSPC development. Wild-type microbiota promote HSPC development by controlling basal inflammatory cytokine expression in kidney niche, and chd8-/- commensals elicit elevated inflammatory cytokines that reduce HSPCs and enhance myeloid differentiation. We identify an Aeromonas veronii strain with immuno-modulatory activities that fails to induce HSPC development in wild-type fish but selectively inhibits kidney cytokine expression and rebalances HSPC development in chd8-/- zebrafish. Our studies highlight the important roles of a balanced microbiome during early HSPC development that ensure proper establishment of lineal precursor for adult hematopoietic system.

CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains.

Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.

Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation.

The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs)1-3. Although recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions4-8, the function of H3.3 in transcription regulation has been less clear9-16. Here, we find that H3.3-specific phosphorylation17-19 stimulates activity of the acetyltransferase p300 in trans, suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Compared with wild-type cells, those lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and impact genome regulation globally, which may help to better understand how mutations in these proteins contribute to human cancers20,21.

Transcription pausing regulates mouse embryonic stem cell differentiation.

The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation.

Dynamic Change of Transcription Pausing through Modulating NELF Protein Stability Regulates Granulocytic Differentiation.

The NELF complex is a metazoan-specific factor essential for establishing transcription pausing. Although NELF has been implicated in cell fate regulation, the cellular regulation of NELF and its intrinsic role in specific lineage differentiation remains largely unknown. Using mammalian hematopoietic differentiation as a model system, here we identified a dynamic change of NELF-mediated transcription pausing as a novel mechanism regulating hematopoietic differentiation. We found a sharp decrease of NELF protein abundance upon granulocytic differentiation and a subsequent genome-wide reduction of transcription pausing. This loss of pausing coincides with activation of granulocyte-affiliated genes and diminished expression of progenitor markers. Functional studies revealed that sustained expression of NELF inhibits granulocytic differentiation, whereas NELF depletion in progenitor cells leads to premature differentiation towards the granulocytic lineage. Our results thus uncover a previously unrecognized regulation of transcription pausing by modulating NELF protein abundance to control cellular differentiation.

Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells.

Therapeutic application of human embryonic stem cells (hESCs) requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs). WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs.