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CEBPA‐IGH, a fusion gene of the immunoglobulin heavy‐chain locus (IGH) and the CCAAT enhancer‐binding protein α (C/EBPα) gene, is recurrently found in B‐ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B‐cell identity due to the inhibition of Pax5, a master regulator of B‐cell differentiation; however, it is not known whether the same mechanism is applicable for B‐ALL development by CEBPA‐IGH. It is known that a full‐length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N‐terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA‐seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B‐cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP‐IGH‐positive ALL (n = 8) compared with other B‐ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP‐qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA‐IGH‐positive ALL and that both isoforms work co‐operatively to achieve it. In the present study, we found that C/EBPα aberrantly expressed in B cells inhibited the function of MEF2 family members by performing RNA‐seq of mRNA of C/EBPα‐transfectants and analyzing RNA‐seq data of 323 clinical samples of ALL. Two isoforms of C/EBPα, p42 and p30, co‐operatively worked for MEF2 inhibition, although it was reported that they worked competitively in AML development.

The stage-specific regulation of sets of enhancers by the 'programming' transcription factor islet 1 regulates the transcription of effector genes during motor neuron differentiation.

The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration.

The divergence of the common dendritic cell progenitor 1 – 3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages 4 , 5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors—such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer 4 , 6 —but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis 7 suggested that Nfil3 acts upstream of Id2 , Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the –165 kb Zeb2 enhancer 8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR–Cas9 targeting showed that these NFIL3–C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3–C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (T H 2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting T H 2 responses to helminths 9 – 11 . Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the –165 kb Zeb2 enhancer. The transcription factor NFIL3 acts antagonistically to C/EBP proteins by binding the Zeb2 enhancer to prevent Zeb2 expression and the development of the conventional type 2 dendritic cell lineage.

This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity.

The preadipocyte-to-adipocyte differentiation (adipogenesis) is a key process in fat mass increase and thus it is regarded as a compelling target for preventing or treating obesity. Of adipogenic hormone receptors, peroxisome proliferator-activated receptor gamma (PPAR γ ) has crucial roles in adipogenesis and lipid accumulation within adipocytes. Here we demonstrate that the NEDD8 (neuronal precursor cell expressed, developmentally downregulated 8)-based post-translation modification (neddylation) of PPAR γ is essential for adipogenesis. During adipogenesis, NEDD8 is robustly induced in preadipocytes and conjugates with PPAR γ , leading to PPAR γ stabilization. When the neddylation process was blocked by NEDD8-targeting siRNAs (or viral vectors) or an inhibitor MLN4924, adipocyte differentiation and fat tissue development were substantially impaired. We also demonstrate that MLN4924 effectively prevents the high-fat diet-induced obesity and glucose intolerance in mice. This study provides a better understanding of how the PPAR γ signaling pathway starts and lasts during adipogenesis and a potential anti-obesity strategy that targets the neddylation of PPAR γ .

Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers.

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

The CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate physiological processes such as energy metabolism, inflammation, cell cycle, and the development and differentiation of several tissues including skin. Recently, a role for C/EBPs in tumor cell proliferation and differentiation has been proposed, but the incomplete characterization in the literature of multiple translational isoforms of these proteins has made interpretation of these roles difficult. Therefore, we have carefully reexamined C/EBP isoform expression in human non-melanoma skin cancers. C/EBPα, C/EBPβ, and C/EBPδ were analyzed histologically in squamous cell carcinomas (SCC). The individual isoforms of C/EBPα and C/EBPβ were examined by immunofluorescent digital imaging, western blotting and DNA binding activity (electrophoretic mobility shift analysis). Expression of all C/EBP family proteins was decreased in SCC tumors. Suppression was greatest for C/EBPα, less for C/EBPβ, and least for C/EBPδ. Western analyses confirmed that C/EBPα p42 and p30 isoforms were decreased. For C/EBPβ, only the abundant full-length isoform (C/EBPβ-1, LAP*, 55 kD) was reduced, whereas the smaller isoforms, C/EBPβ-2 (LAP, 48 kD) and C/EBPβ-3 (LIP, 20 kD), which are predominantly nuclear, were significantly increased in well- and moderately-differentiated SCC (up to 14-fold for C/EBPβ-3). These elevations correlated with increases in PCNA, a marker of proliferation. Although C/EBPβ displayed increased post-translational modifications in SCC, phosphorylation of C/EBPβ-1 (Thr 235) was not altered. C/EBP-specific DNA binding activity in nuclear and whole-cell extracts of cultured cells and tumors was predominantly attributable to C/EBPβ. In summary, two short C/EBPβ isoforms, C/EBPβ-2 and C/EBPβ-3, represent strong candidate markers for epithelial skin malignancy, due to their preferential expression in carcinoma versus normal skin, and their strong correlation with tumor proliferation.

An atypical monocyte with partial granulocyte characteristics is identified and shown to be critical for the development of fibrosis. An immunological cell type inducing liver fibrosis In this study, Shizuo Akira and colleagues identify a previously unknown monocyte–granulocyte hybrid cell type as being critical to the development of bleomycin-induced pulmonary fibrosis in mice, the most commonly used experimental study model of human lung fibrosis. The cells, termed segregated-nucleus-containing atypical monocytes (SatMs), are differentiated from committed progenitor cells under the control of the transcription factor C/EBPβ. The authors speculate that SatMs, and other reported 'disorder-specific monocyte/macrophage subtypes' corresponding to certain diseases, might be investigated as highly specific therapeutic targets. Monocytes and macrophages comprise a variety of subsets with diverse functions 1 , 2 , 3 , 4 , 5 . It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages 6 , 7 , 8 . However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1 + Msr1 + Ly6C − F4/80 − Mac1 + monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb −/− haematopoietic cells. Adoptive transfer of SatM into Cebpb −/− mice resulted in fibrosis. Notably, SatM are derived from Ly6C − FcεRI + granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C − FcεRI + granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.