Co-expression of PADI isoforms during progenitor differentiation enables functional diversity.

Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted Padi3α and Padi3ß isoforms in hair follicles and describe expression of Padi2ß, a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. Padi2ß transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2ß. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao et al. 2019 Cell Rep. 27, 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2ß inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize Padi3α and Padi3ß to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed in vitro, PADI3α and PADI3ß are enriched in the cytoplasm and precipitate together. Whereas PADI3ß protein stability is low and PADI3ß fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3ß. We propose that PADI3ß modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of Padi2 and Padi3 isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

ID1 and CEBPA coordinate epidermal progenitor cell differentiation.

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here, we report that the transcriptional regulator ID1 is enriched in mouse basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests that ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and establish TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1, and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.

A Hairy Cituation - PADIs in Regeneration and Alopecia.

In this Review article, we focus on delineating the expression and function of Peptidyl Arginine Delminases (PADIs) in the hair follicle stem cell lineage and in inflammatory alopecia. We outline our current understanding of cellular processes influenced by protein citrullination, the PADI mediated posttranslational enzymatic conversion of arginine to citrulline, by exploring citrullinomes from normal and inflamed tissues. Drawing from other stem cell lineages, we detail the potential function of PADIs and specific citrullinated protein residues in hair follicle stem cell activation, lineage specification and differentiation. We highlight PADI3 as a mediator of hair shaft differentiation and display why mutations in PADI3 are linked to human alopecia. Furthermore, we propose mechanisms of PADI4 dependent fine-tuning of the hair follicle lineage progression. Finally, we discuss citrullination in the context of inflammatory alopecia. We present how infiltrating neutrophils establish a citrullination-driven self-perpetuating proinflammatory circuitry resulting in T-cell recruitment and activation contributing to hair follicle degeneration. In summary, we aim to provide a comprehensive perspective on how citrullination modulates hair follicle regeneration and contributes to inflammatory alopecia.

A Combinatorial CRISPR-Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment.

Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.

Histone demethylase JMJD2B/KDM4B regulates transcriptional program via distinctive epigenetic targets and protein interactors for the maintenance of trophoblast stem cells.

Trophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B-TFAP2C-LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C-LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer-promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets.

Murine Mesenchymal Stromal Cells Retain Biased Differentiation Plasticity Towards Their Tissue of Origin.

Mesenchymal stromal/stem cells (MSCs) reside in many human tissues and comprise a heterogeneous population of cells with self-renewal and multi-lineage differentiation potential, making them useful in regenerative medicine. It remains inconclusive whether MSCs isolated from different tissue sources exhibit variations in biological features. In this study, we derived MSCs from adipose tissue (AT-MSC) and compact bone (CB-MSC). We found that early passage of MSCs was readily expandable ex vivo, whereas the prolonged culture of MSCs showed alteration of cell morphology to fibroblastoid and reduced proliferation. CB-MSCs and AT-MSCs at passage 3 were CD29+, CD44+, CD105+, CD106+, and Sca-1+; however, passage 7 MSCs showed a reduction of MSC markers, indicating loss of stem cell population after prolonged culturing. Strikingly, CB-MSC was found more efficient at undergoing osteogenic differentiation, while AT-MSC was more efficient to differentiate into adipocytes. The biased differentiation pattern of MSCs from adipogenic or osteogenic tissue source was accompanied by preferential expression of the corresponding lineage marker genes. Interestingly, CB-MSCs treated with DNA demethylation agent 5-azacytidine showed enhanced osteogenic and adipogenic differentiation, whereas the treated AT-MSCs are less competent to differentiate. Our results suggest that the epigenetic state of MSCs is associated with the biased differentiation plasticity towards its tissue of origin, proposing a mechanism related to the retention of epigenetic memory. These findings facilitate the selection of optimal tissue sources of MSCs and the ex vivo expansion period for therapeutic applications.

Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells.

MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.