Polycomb repressive complex 2 accessory factors: rheostats for cell fate decision?

Epigenetic reprogramming during development is key to cell identity and the activities of the Polycomb repressive complexes are vital for this process. We focus on polycomb repressive complex 2 (PRC2), which catalyzes H3K27me1/2/3 and safeguards cellular integrity by ensuring proper gene repression. Notably, various accessory factors associate with PRC2, strongly influencing cell fate decisions, and their deregulation contributes to various illnesses. Yet, the exact role of these factors during development and carcinogenesis is not fully understood. Here, we present recent progress toward addressing these points and an analysis of the expression levels of PRC2 accessory factors in various tissues and developmental stages to highlight their abundance and roles. Last, we evaluate their contribution to cancer-specific phenotypes, providing insight into novel anticancer therapies.

Functional Properties of Rice Bran Proteins Extracted from Low-Heat-Treated Defatted Rice Bran.

Rice bran is rich in proteins with high nutritional values. However, current protein extraction methods from rice bran are greatly limited by their low yield. Therefore, in this study, we aimed to develop a feasible method to extract rice bran protein (RBP) of high purity and quality. We prepared RBP using low-heat-treated defatted rice bran (LDRB) and analyzed its functional properties. The protein solubility of LDRB increased from 25.4% to 56% upon increasing the pH level and was more than double that of heat-stabilized defatted rice bran. RBP prepared from LDRB had good functional properties, comparable to those of soy proteins. The emulsifying capacities of RBP were 424 ± 14 mL/g at pH 4 and 530 ± 21 mL/g at pH 7.0. Under acidic conditions, RBP showed a better emulsifying capacity than soy proteins (262 ± 1 mL/g at pH 4). RPB showed water-binding and oil-absorption capacities of 270 ± 35 g/100 g and 268 ± 30 g/100 g, respectively. Moreover, RBP showed better foaming capacity (610% vs. 590%) and foam stability (83% vs. 4%) than soy proteins; however, it lacked gelling properties. This study demonstrated that RBP is a potential new protein source in the food industry.

Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells.

Inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate (IP) metabolism, is a pleiotropic signaling factor involved in major biological events, including transcriptional control. In the yeast, IPMK and its IP products promote the activity of the chromatin remodeling complex SWI/SNF, which plays a critical role in gene expression by regulating chromatin accessibility. However, the direct link between IPMK and chromatin remodelers remains unclear, raising the question of how IPMK contributes to transcriptional regulation in mammals. By employing unbiased screening approaches and in vivo/in vitro immunoprecipitation, here we demonstrate that mammalian IPMK physically interacts with the SWI/SNF complex by directly binding to SMARCB1, BRG1, and SMARCC1. Furthermore, we identified the specific domains required for IPMK-SMARCB1 binding. Notably, using CUT&RUN and ATAC-seq assays, we discovered that IPMK co-localizes with BRG1 and regulates BRG1 localization as well as BRG1-mediated chromatin accessibility in a genome-wide manner in mouse embryonic stem cells. Together, these findings show that IPMK regulates the promoter targeting of the SWI/SNF complex, thereby contributing to SWI/SNF-meditated chromatin accessibility, transcription, and differentiation in mouse embryonic stem cells.

Comparative characterization of 3D chromatin organization in triple-negative breast cancers.

Triple-negative breast cancer (TNBC) is a malignant cancer subtype with a high risk of recurrence and an aggressive phenotype compared to other breast cancer subtypes. Although many breast cancer studies conducted to date have investigated genetic variations and differential target gene expression, how 3D chromatin architectures are reorganized in TNBC has been poorly elucidated. Here, using in situ Hi-C technology, we characterized the 3D chromatin organization in cells representing five distinct subtypes of breast cancer (including TNBC) compared to that in normal cells. We found that the global and local 3D architectures were severely disrupted in breast cancer. TNBC cell lines (especially BT549 cells) showed the most dramatic changes relative to normal cells. Importantly, we detected CTCF-dependent TNBC-susceptible losses/gains of 3D chromatin organization and found that these changes were strongly associated with perturbed chromatin accessibility and transcriptional dysregulation. In TNBC tissue, 3D chromatin disorganization was also observed relative to the 3D chromatin organization in normal tissues. We observed that the perturbed local 3D architectures found in TNBC cells were partially conserved in TNBC tissues. Finally, we discovered distinct tissue-specific chromatin loops by comparing normal and TNBC tissues. In this study, we elucidated the characteristics of the 3D chromatin organization in breast cancer relative to normal cells/tissues at multiple scales and identified associations between disrupted structures and various epigenetic features and transcriptomes. Collectively, our findings reveal important 3D chromatin structural features for future diagnostic and therapeutic studies of TNBC.

CHD4 Conceals Aberrant CTCF-Binding Sites at TAD Interiors by Regulating Chromatin Accessibility in Mouse Embryonic Stem Cells.

CCCTC-binding factor (CTCF) critically contributes to 3D chromatin organization by determining topologically associated domain (TAD) borders. Although CTCF primarily binds at TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking the putative CTCF-binding sites remains largely elusive. Here, we show that the ATP-dependent chromatin remodeler, chromodomain helicase DNA-binding 4 (CHD4), regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 short interspersed nuclear elements (SINEs) in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible and aberrant CTCF recruitment occurs within TADs, resulting in disorganization of local TADs. RNA-binding intrinsically disordered domains (IDRs) of CHD4 are required to prevent this aberrant CTCF binding, and CHD4 is critical for the repression of B2 SINE transcripts. These results collectively reveal that a CHD4-mediated mechanism ensures appropriate CTCF binding and associated TAD organization in mESCs.

The chromatin remodeler Ino80 mediates RNAPII pausing site determination.

BACKGROUND: Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. RESULTS: Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. CONCLUSIONS: Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors.

Pediatric brain tumors are highly associated with epileptic seizures1. However, their epileptogenic mechanisms remain unclear. Here, we show that the oncogenic BRAF somatic mutation p.Val600Glu (V600E) in developing neurons underlies intrinsic epileptogenicity in ganglioglioma, one of the leading causes of intractable epilepsy2. To do so, we developed a mouse model harboring the BRAFV600E somatic mutation during early brain development to reflect the most frequent mutation, as well as the origin and timing thereof. Therein, the BRAFV600E mutation arising in progenitor cells during brain development led to the acquisition of intrinsic epileptogenic properties in neuronal lineage cells, whereas tumorigenic properties were attributed to high proliferation of glial lineage cells. RNA sequencing analysis of patient brain tissues with the mutation revealed that BRAFV600E-induced epileptogenesis is mediated by RE1-silencing transcription factor (REST), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy. Moreover, we found that seizures in mice were significantly alleviated by an FDA-approved BRAFV600E inhibitor, vemurafenib, as well as various genetic inhibitions of Rest. Accordingly, this study provides direct evidence of a BRAF somatic mutation contributing to the intrinsic epileptogenicity in pediatric brain tumors and suggests that BRAF and REST could be treatment targets for intractable epilepsy.

Biogenesis and regulation of the let-7 miRNAs and their functional implications.

The let-7 miRNA was one of the first miRNAs discovered in the nematode, Caenorhabditis elegans, and its biological functions show a high level of evolutionary conservation from the nematode to the human. Unlike in C. elegans, higher animals have multiple isoforms of let-7 miRNAs; these isoforms share a consensus sequence called the 'seed sequence' and these isoforms are categorized into let-7 miRNA family. The expression of let-7 family is required for developmental timing and tumor suppressor function, but must be suppressed for the self-renewal of stem cells. Therefore, let-7 miRNA biogenesis must be carefully controlled. To generate a let-7 miRNA, a primary transcript is produced by RNA polymerase II and then subsequently processed by Drosha/DGCR8, TUTase, and Dicer. Because dysregulation of let-7 processing is deleterious, biogenesis of let-7 is tightly regulated by cellular factors, such as the RNA binding proteins, LIN28A/B and DIS3L2. In this review, we discuss the biological functions and biogenesis of let-7 miRNAs, focusing on the molecular mechanisms of regulation of let-7 biogenesis in vertebrates, such as the mouse and the human.

Nutritional quality of rice bran protein in comparison to animal and vegetable protein.

Rice bran protein (RBP) was prepared by alkali extraction and isoelectric precipitation from defatted rice bran. The protein quality of RPB was evaluated and compared to two vegetable proteins [soy protein (ISP) and rice endosperm protein (REP)] and two animal proteins [whey protein (WPI) and casein]. RPB contained 74.93% of protein and its pepsin digestibility and KOH solubility were 89.8% and 91.5%, respectively. In Sprague-Dawley rats, RBP showed protein efficiency ratio, net protein ratio, net protein utilisation, and biological value of 2.39, 3.77, 70.7, and 72.6, which were comparable to the qualities of animal proteins. The true digestibility of RBP (94.8%) was significantly higher than that of REP (90.8%), ISP (91.7%) and WPI (92.8%) and the same as that of casein. Protein digestibility corrected amino acid score (PDCAAS) of RBP was 0.90. These results suggest that rice bran protein appears to be a promising protein source with good biological values and digestibility.