Opisthorchis viverrini infection is endemic in the lower Mekong subregion. The liver is an organ that worms are drawn to and cause damage. However, the immune-related susceptibility in the liver is poorly understood. In this study, we investigated T helper (Th) cell responses in the liver of BALB/c mice and golden Syrian hamsters during 2-28 days post-infection (DPI). We found that Th cell responses were distinct between mice and hamsters in terms of dynamics and polarization. Mice exhibited the early induction of Th1, Th2, Th17, and regulatory T (Treg) cells responses after the presence of O. viverrini worms at 2 DPI. In hamsters, the late induction of Th1/Th17, downregulation of Th2/Treg responses and early elevation of suppressive cytokine interleukin (IL)-10 were found together with swift reduction of Th cell numbers. Interestingly, expressions of IL-4 (Th2 functional cytokine) and Foxp3 (Treg lineage) were completely different between mice and hamsters which elevated in mice but suppressed in hamsters. These results suggest that early induction and well-regulation are related to host resistance. In contrast, late induction of Th cell response might allow immature worms to develop in the host. Our findings provide a greater understanding in Th cell response-related susceptibility in O. viverrini infection which would be targeting immunity for the development of immune-based intervention such as vaccine.

Engineered promoters are key components that allow engineered expression of genes in the cell-factory design. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Engineered promoter variants (EPVs) of naturally occurring promoters (NOPs) can be designed using metabolic engineering strategies and synthetic biology tools if the genes encoding the activating transcription factors (TFs) exist in the genome and are expressed and synthesized at non-limiting concentrations within the cell. The hybrid-architectured EPV design method targets an essential and predetermined part of the general transcription machinery. That is cis-acting DNA site(s) in coordination with the trans-acting factor(s) that must bind for the regulated transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, enabling the engineering of binding of master regulator TF(s). The method aims to generate EPVs that combine the advantages of being an exceptional stronger EPV(s) than the NOPs and permit \"green-and-clean production\" on a non-toxic carbon source, such as ethanol or glucose. By introducing our recent work on the engineering of ADH2 hybrid-promoter architectures to enhance recombinant protein expression on ethanol, we provide the method and protocol for the design of ADH2 hybrid-promoter architectures that can be adapted to other promoters in different substrate utilization pathways in Pichia pastoris (syn. Komagataella phaffii), as well as in other yeasts.

Hybrid-architectured promoter design to deregulate expression in yeast under modulating power of carbon sources involves replacing native cis-acting DNA sequence(s) with de novo synthetic tools in coordination with master regulator transcription factor (TF) to alter crosstalk between signaling pathways, and consequently, transcriptionally rewire the expression. Hybrid-promoter architectures can be designed to mimic native promoter architectures in yeast\'s preferred carbon source utilization pathway. The method aims to generate engineered promoter variants (EPVs) that combine the advantages of being an exceptionally stronger EPV(s) than the naturally occurring promoters and permit \"green-and-clean\" production on a non-toxic carbon source. To implement the method, a predetermined essential part of the general transcription machinery is targeted. This targeting involves cis-acting DNA sequences to be replaced with synthetic cis-acting DNA sites in coordination with the targeted TF that must bind for transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, which enable the engineering of binding of master regulator TF(s). By introducing our recent work on the engineering of Pichia pastoris (syn. Komagataella phaffii) alcohol oxidase 1 (AOX1) hybrid-promoter architectures, we provide the method and protocol for the hybrid-architectured EPV design to deregulate expression in yeast. The method can be adapted to other promoters in different substrate utilization pathways in P. pastoris, as well as in other yeasts.

PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Cell fate conversion by overexpressing defined factors is a powerful tool in regenerative medicine. However, identifying key factors for cell fate conversion requires laborious experimental efforts; thus, many of such conversions have not been achieved yet. Nevertheless, cell fate conversions found in many published studies were incomplete as the expression of important gene sets could not be manipulated thoroughly. Therefore, the identification of master transcription factors for complete and efficient conversion is crucial to render this technology more applicable clinically. In the past decade, systematic analyses on various single-cell and bulk OMICs data have uncovered numerous gene regulatory mechanisms, and made it possible to predict master gene regulators during cell fate conversion. By virtue of the sparse structure of master transcription factors and the group structure of their simultaneous regulatory effects on the cell fate conversion process, this study introduces a novel computational method predicting master transcription factors based on group sparse optimization technique integrating data from multi-OMICs levels, which can be applicable to both single-cell and bulk OMICs data with a high tolerance of data sparsity. When it is compared with current prediction methods by cross-referencing published and validated master transcription factors, it possesses superior performance. In short, this method facilitates fast identification of key regulators, give raise to the possibility of higher successful conversion rate and in the hope of reducing experimental cost.

Obesity predisposes individuals to multiple cardiometabolic disorders, including type 2 diabetes (T2D). As body mass index (BMI) cannot reliably differentiate fat from lean mass, the metabolically detrimental abdominal obesity has been estimated using waist-hip ratio (WHR). Waist-hip ratio adjusted for body mass index (WHRadjBMI) in turn is a well-established sex-specific marker for abdominal fat and adiposity, and a predictor of adverse metabolic outcomes, such as T2D. However, the underlying genes and regulatory mechanisms orchestrating the sex differences in obesity and body fat distribution in humans are not well understood.
We searched for genetic master regulators of WHRadjBMI by employing integrative genomics approaches on human subcutaneous adipose RNA sequencing (RNA-seq) data (n ~ 1400) and WHRadjBMI GWAS data (n ~ 700,000) from the WHRadjBMI GWAS cohorts and the UK Biobank (UKB), using co-expression network, transcriptome-wide association study (TWAS), and polygenic risk score (PRS) approaches. Finally, we functionally verified our genomic results using gene knockdown experiments in a human primary cell type that is critical for adipose tissue function.
Here, we identified an adipose gene co-expression network that contains 35 obesity GWAS genes and explains a significant amount of polygenic risk for abdominal obesity and T2D in the UKB (n = 392,551) in a sex-dependent way. We showed that this network is preserved in the adipose tissue data from the Finnish Kuopio Obesity Study and Mexican Obesity Study. The network is controlled by a novel adipose master transcription factor (TF), TBX15, a WHRadjBMI GWAS gene that regulates the network in trans. Knockdown of TBX15 in human primary preadipocytes resulted in changes in expression of 130 network genes, including the key adipose TFs, PPARG and KLF15, which were significantly impacted (FDR < 0.05), thus functionally verifying the trans regulatory effect of TBX15 on the WHRadjBMI co-expression network.
Our study discovers a novel key function for the TBX15 TF in trans regulating an adipose co-expression network of 347 adipose, mitochondrial, and metabolically important genes, including PPARG, KLF15, PPARA, ADIPOQ, and 35 obesity GWAS genes. Thus, based on our converging genomic, transcriptional, and functional evidence, we interpret the role of TBX15 to be a main transcriptional regulator in the adipose tissue and discover its importance in human abdominal obesity.

Kaposi\'s sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas.IMPORTANCE Kaposi\'s sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a member of the steroid/thyroid hormone receptor superfamily, but its ligand has not yet been identified. Little is known about the role of the COUP-TFII nuclear receptor in cancer cells. In this study, we mapped the cistrome of COUP-TFII in three different cancer cells, namely breast cancer cells (MCF-7), myelogenous leukaemia cells (K562) and liver cancer cells (HepG2) using publicly available ChIP-seq data. Our results show that COUP-TFII co-localises with master transcription factors (TFs) in a cell-specific manner such as estrogen receptor alpha in MCF-7, hepatocyte nuclear factor alpha in HepG2, and GATA-binding factor in K562, while the shared, non-specific COUP-TFII binding sites are co-occupied by CTCF. We identified chromatin environments for these COUP-TFII and master TF co-bound sites together with COUP-TFII and CTCF co-bound sites. Our results show that COUP-TFII and master TF co-bound sites are marked with active enhancer specific histone modifications (H3K27ac and H3K4me1), while COUP-TFII and CTCF co-bound sites reveal active promoter specific histone marks (H3K27ac and H3K4me3). These results describe the genomic context and role of COUP-TFII in the cell-type specific transcriptional programs. Furthermore, we report that the VEGFA gene regulated by shared COUP-TFII and CTCF co-bound regulatory elements is involved in long-range looping in a cell-type-independent manner. These findings provide a genomic insight into the regulation and angiogenic role of COUP-TFII.

GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.