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The impressive conversational and programming abilities of ChatGPT make it an attractive tool for facilitating the education of bioinformatics data analysis for beginners. In this study, we proposed an iterative model to fine‐tune instructions for guiding a chatbot in generating code for bioinformatics data analysis tasks. We demonstrated the feasibility of the model by applying it to various bioinformatics topics. Additionally, we discussed practical considerations and limitations regarding the use of the model in chatbot‐aided bioinformatics education.

Articular cartilage has a limited capacity to self-heal once damaged. Tissue-specific stem cells are a solution for cartilage regeneration; however, ex vivoexpansion resulting in cell senescence remains a challenge as a large quantity of high-quality tissue-specific stem cells are needed for cartilage regeneration. Our previous report demonstrated that decellularized extracellular matrix (dECM) deposited by human synovium-derived stem cells (SDSCs), adipose-derived stem cells (ADSCs), urine-derived stem cells (UDSCs), or dermal fibroblasts (DFs) provided an ex vivosolution to rejuvenate human SDSCs in proliferation and chondrogenic potential, particularly for dECM deposited by UDSCs. To make the cell-derived dECM (C-dECM) approach applicable clinically, in this study, we evaluated ex vivorejuvenation of rabbit infrapatellar fat pad-derived stem cells (IPFSCs), an easily accessible alternative for SDSCs, by the abovementioned C-dECMs, in vivoapplication for functional cartilage repair in a rabbit osteochondral defect model, and potential cellular and molecular mechanisms underlying this rejuvenation. We found that C-dECM rejuvenation promoted rabbit IPFSCs' cartilage engineering and functional regeneration in both ex vivoand in vivomodels, particularly for the dECM deposited by UDSCs, which was further confirmed by proteomics data. RNA-Seq analysis indicated that both mesenchymal-epithelial transition (MET) and inflammation-mediated macrophage activation and polarization are potentially involved in the C-dECM-mediated promotion of IPFSCs’ chondrogenic capacity, which needs further investigation.

Metastatic breast cancer causes most breast cancer-associated deaths, especially in triple negative breast cancers (TNBC). The metastatic drivers of TNBCs are still poorly understood, and effective treatment non-existent. Here we reveal that the presence of Aurora-A Kinase (AURKA) in the nucleus and metastatic dissemination are molecularly connected through HIF1 (Hypoxia-Inducible Factor-1) signaling. Nuclear AURKA activates transcription of “hypoxia-induced genes” under normoxic conditions (pseudohypoxia) and without upregulation of oxygen-sensitive HIF1A subunit. We uncover that AURKA preferentially binds to HIF1B and co-localizes with the HIF complex on DNA. The mass-spectrometry analysis of the AURKA complex further confirmed the presence of CBP and p300 along with other TFIIB/RNApol II components. Importantly, the expression of multiple HIF-dependent genes induced by nuclear AURKA (N-AURKA), including migration/invasion, survival/death, and stemness, promote early cancer dissemination. These results indicate that nuclear, but not cytoplasmic, AURKA is a novel driver of early metastasis. Analysis of clinical tumor specimens revealed a correlation between N-AURKA presence and decreased patient survival. Our results establish a mechanistic link between two critical pathways in cancer metastasis, identifying nuclear AURKA as a crucial upstream regulator of the HIF1 transcription complex and a target for anti-metastatic therapy.

The transcriptional repressor Hairy Enhancer of Split 1 (HES1) plays an essential role in the development of many organs by promoting the maintenance of stem/progenitor cells, controlling the reversibility of cellular quiescence, and regulating both cell fate decisions. Deletion of Hes1in mice results in severe defects in multiple organs and is lethal in late embryogenesis. Here we have investigated the role of HES1 in hematopoiesis using a hematopoietic lineage‐specific Hes1knockout mouse model. We found that while Hes1is dispensable for steady‐state hematopoiesis, Hes1‐deficient hematopoietic stem cells (HSCs) undergo exhaustion under replicative stress. Loss of Hes1upregulates the expression of genes involved in PPARγ signaling and fatty acid metabolism pathways, and augments fatty acid oxidation (FAO) in Hes1f/fVav1CreHSCs and progenitors. Functionally, PPARγ targeting or FAO inhibition ameliorates the repopulating defects of Hes1f/fVav1CreHSCs through improving quiescence in HSCs. Lastly, transcriptome analysis reveals that disruption of Hes1in hematopoietic lineage alters expression of genes critical to HSC function, PPARγ signaling, and fatty acid metabolism. Together, our findings identify a novel role of HES1 in regulating stress hematopoiesis and provide mechanistic insight into the function of HES1 in HSC maintenance. While Hes1 is dispensable for steady‐state hematopoiesis, Hes1‐deficient HSCs undergo exhaustion under replicative stress. Deletion of Hes1 deregulates genes in PPARγ signaling and fatty acid oxidation (FAO), and augments FAO in Hes1f/fVav1Crehematopoietic stem cells (HSCs) and progenitors. Mechanistically, HES1 plays important role in stress hematopoiesis by regulating genes in HSC function, PPARγ signaling and fatty acid metabolism pathways.

T follicular helper (Tfh) cells express transcription factor BCL-6 and cytokine IL-21. Mature Tfh cells are also capable of producing IFN-γ without expressing the Th1 transcription factor T-bet. Whether this IFN-γ–producing Tfh population represents a unique Tfh subset with a distinct differentiation pathway is poorly understood. By using T-bet fate–mapping mouse strains, we discovered that almost all the IFN-γ–producing Tfh cells have previously expressed T-bet and express high levels of NKG2D. DNase I hypersensitivity analysis indicated that the Ifng gene locus is partially accessible in this “ex–T-bet” population with a history of T-bet expression. Furthermore, multicolor tissue imaging revealed that the ex–T-bet Tfh cells found in germinal centers express IFN-γ in situ. Finally, we found that IFN-γ–expressing Tfh cells are absent in T-bet–deficient mice, but fully present in mice with T-bet deletion at late stages of T cell differentiation. Together, our findings demonstrate that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in this Th1-like Tfh cell subset.

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1–3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3–6and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9that may be related to heterogeneity in chromatin accessibility10–12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.

Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+T cells revealed substantial reorganization of enhancer–promoter interactions associated with changes in gene expression after T cell receptor stimulation.

Type 1 T helper (Th1) cells play a critical role in host defense against intracellular pathogens and in autoimmune diseases by producing a key inflammatory cytokine interferon (IFN)–γ; some Th1 cells can also be antiinflammatory through producing IL-10. However, the molecular switch for regulating the differentiation of inflammatory and antiinflammatory Th1 cells is still elusive. Here, we show that Bhlhe40-deficient CD4 Th1 cells produced less IFN-γ but substantially more IL-10 than wild-type Th1 cells both in vitro and in vivo. Bhlhe40-mediated IFN-γ production was independent of transcription factor T-bet regulation. Mice with conditional deletion of Bhlhe40 in T cells succumbed to Toxoplasma gondii infection, and blockade of IL-10 signaling during infection rescued these mice from death. Thus, our results demonstrate that transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and antiinflammatory Th1 cells.

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein–protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer’s patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

MLL4 is an essential subunit of the histone H3 Lys4 (H3K4)-methylation complexes. We found that MLL4 deficiency compromised the development of regulatory T cells (Tregcells) and resulted in a substantial decrease in monomethylated H3K4 (H3K4me1) and chromatin interaction at putative gene enhancers, a considerable portion of which were not direct targets of MLL4 but were enhancers that interacted with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the enhancers not bound by MLL4 correlated with MLL4 binding at distant interacting regions. Deletion of an upstream MLL4-binding site diminished the abundance of H3K4me1 at the regulatory elements of the gene encoding the transcription factor Foxp3 that were looped to the MLL4-binding site and compromised both the thymic differentiation and the inducible differentiation of Tregcells. We found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping, which identifies a previously unknown mechanism for regulating the T cell enhancer landscape and affecting Tregcell differentiation.

Abstract ID 27846

Our method for analyzing histone modifications, scChIC-seq (single-cell chromatin immunocleavage sequencing), involves targeting of the micrococcal nuclease (MNase) to a histone mark of choice by tethering to a specific antibody. Cleaved target sites are then selectively PCR amplified. We show that scChIC-seq reliably detects H3K4me3 and H3K27me3 target sites in single human white blood cells. The resulting data are used for clustering of blood cell types.

Type 2 T helper (Th2) cells and group 2 innate lymphoid cells (ILC2s) provide protection against helminth infection and are involved in allergic responses. However, their relative importance and crosstalk during type 2 immune responses are still controversial. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that interleukin (IL)-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins.

The transcription factor GATA-3 is indispensable for the development of all innate lymphoid cells (ILCs) that express the interleukin 7 receptor α-chain (IL-7Rα). However, the function of low GATA-3 expression in committed group 3 ILCs (ILC3 cells) has not been identified. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served a critical function in the development of the NKp46+ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Among NKp46+ILC3 cells, although GATA-3 positively regulated genes specific to the NKp46+ILC3 subset, it negatively regulated genes specific to lymphoid tissue–inducer (LTi) or LTi-like ILC3 cells. Furthermore, GATA-3 was required for IL-22 production in both ILC3 subsets. Thus, despite its low expression, GATA-3 was critical for the homeostasis, development and function of ILC3 subsets.

Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy, and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers, and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a general facilitator that generates access for a variety of complexes, both activating and repressive.

Change history: In Fig. 1c of this Letter, the two graphs were duplicates. The right panel of Fig. 1c has been corrected online.

Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function.

No relevant conflicts of interest to declare.

Abstract 273