Down syndrome (DS) is caused by the triplication of chromosome 21, but science is still investigating the precise mechanisms by which this results in the various phenotypes, such as anatomical abnormalities, intellectual deficits, and early development of Alzheimer’s disease (AD). The global changes in transcriptional activity and the altered expression of genes not transcribed from chromosome 21 point to changes to the epigenetic landscape. One of the candidate genes for this global gene dysregulation is High Mobility Group Nucleosome Binding Domain 1 (HMGN1) which is triplicated in DS. While investigating DS-associated B-cell acute lymphoblastic leukemia (B-ALL), researchers found the triplication of HMGN1 alone led to many of the same transcriptional and phenotypic changes that marked DS-associated B-cells from a mouse model with all 31 genes orthologous to human chromosome 21 genes triplicated. Amongst the pathways most affected by triplication, enrichment was greatest for targets of the Polycomb Repressive Complex 2 (PRC2) and sites of the transcriptionally repressive mark it catalyzes, H3K27me3. HMGN1 instead, promotes transcriptional activation and its overexpression leads to a global increase in RNA transcript levels. Therefore, overexpression of HMGN1 in DS may cause an increase in transcriptional activity and prevent the silencing of genes normally silenced by PRC2, with downstream effects on neurogenesis and gliogenesis, abnormal cellular migration, and deviant developmental timing that result in known DS phenotypes. With this hypothesis, we first wanted to quantify the levels of acetylation versus methylation at H3K27 in trisomy 21 induced pluripotent stem (iPS) cell-derived cellular models: neural progenitor cells (NPCs) and cortical organoids and to determine if there are measurable differences between the genotypes. We found a decrease in H3K27me3 in 130-day-old organoids, but not in NPCs. No changes were detected in the levels of H3K27ac. With the high comorbidity between DS and AD, and changes to the epigenome found in both diseases, we wondered whether there were specific alterations at H3K27 in DS-AD. To determine this, we performed an analysis of human postmortem brain tissue from individuals with DS-AD, AD, and control and quantified H3K27me3 and H3K27ac marks. Our data indicated that there are marginally significant changes in H3K27me3 that are unique to DS-AD as compared to control and AD samples. Encouraged by this data, we next measured gene expression levels of specific PRC2 target genes increased in trisomy. Our goal was to identify the causative relationship between the increased expression of HMGN1 in trisomy and upregulation of specific PRC2 target genes with known brain-related functions. We found that enhanced expression of particular PRC2 target genes in trisomic cells could be normalized with the short-hairpin RNA (shRNA)-induced knockdown of HMGN1 expression in trisomic NPCs. This implicates HMGN1 overexpression in DS in the dysregulation and overexpression of particular genes involved in morphogenesis, neurogenesis, neuronal migration, apoptosis, and cell viability through the antagonism of the PRC2 activity. We provided novel evidence for a possible mechanism for the cellular, molecular, and transcriptomic changes originating from the triplication of HMGN1 that can potentially lead to DS-related phenotypes such as intellectual disability and AD-related pathology. Furthermore, our findings suggest a possible therapeutic avenue to mitigate these phenotypes by regulating HMGN1 expression. Taken together, our work is the first to causatively connect HMGN1-induced epigenetic changes to DS-related brain cell phenotypes and to point out to a potential approach for correcting them. .

Indiana University-Purdue University Indianapolis (IUPUI) / Hepatic fibrosis is a cellular repair mechanism that is initiated upon prolonged damage to the liver, resulting in an accumulation of excess extracellular matrix. This eventually leads to the formation of scar tissue, which disrupts the hepatic architecture and causes liver dysfunction. Hepatic stellate cells (HSCs) play a major role in hepatic fibrosis. However, the molecular mechanisms remain incompletely understood. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ or WWTR1), key players of the Hippo pathway, have been implicated in the liver fibrosis, but the HSC-specific functions of YAP and TAZ are largely unclear. Here we have identified Sirtuin 6 (SIRT6), an NAD+ dependent deacetylase, as a key epigenetic regulator in the protection against hepatic fibrosis by suppressing the YAP/TAZ activity. SIRT6 has been previously implicated in the regulation of the canonical transforming growth factor β (TGFβ)-SMAD3 pathway. This study has revealed the significant contribution of the non-canonical pathways including the Hippo pathway to the development of hepatic fibrosis. HSC-specific Sirt6 deficient mice developed severe fibrosis when fed a high-fat-cholesterol-cholate diet compared to their wild-type counterparts. YAP became more active in the SIRT6-deficient HSCs. Expression of the YAP/TAZ downstream genes like CTGF, CYR61 and ANKRD1 were elevated in the SIRT6-deficient HSCs. Biochemical and mutagenic analyses have revealed that SIRT6 deacetylates YAP and TAZ at key lysine residues and reprograms the composition of the TEA domain transcription factor complex to suppress the YAP/TAZ function in the hepatic fibrogenesis.

Male fertility, ability to fertilize and activate the egg and support early embryo development, is crucial for mammalian reproduction and development. Testis specific histone 2B (TH2B) of sperm, protamines (PRM1/2), and posttranslational modifications of histone 3 (H3K27me3 and H3k27ac) are involved in spermatogenesis and male fertility. However, molecular and cellular mechanisms by which TH2B regulates histone to protamine replacement is poorly defined. Immunocytochemistry, western blotting, flow cytometry, computer-assisted sperm analysis (CASA) and bioinformatic approaches were applied to analyze sperm from Holstein bulls with different in vivo fertility. Results from the immunocytochemistry experiments showed that while TH2B and H3K27me3 were localized predominantly at the equatorial and post acrosomal (localized as a crown around the sperm head) parts, respectively. The H3K27ac was also detectable in the bovine sperm head. Signal intensities of TH2B (mean ± SEM) were higher in sperm from the low fertility bulls (220.56 ± 9.20) as compared to those from the high fertility bulls (198.39 ± 10.0). Signal intensities of H3K27me3 (16.25 ± 1.69) were significantly different than those of H3K27ac (4.74 ± 0.88) in bull spermatozoa. Using the bioinformatic tools, including Clustal Omega, Cytoscape, Emboss Dotmatcher, InterProScan, and STRING, we demonstrated that TH2B has the conserved histone H2B domain which has a strong association with proteins involved in chromosome organization and histone ubiquitination. Intensities of PRM1 and PRM2 were significantly associated with one another (p < 0.0001), but neither were significantly associated with fertility. Results from CASA revealed significant differences between high and low fertility bulls regarding average sperm pathway velocity, amplitude of lateral head displacement and straightness (p < 0.05). The interacting proteins of H3 are involved in subcellular processes such as regulation of H3K27 methylation, nucleosome assembly, regulation of DNA replication, and chromatin assembly. These results are significant because they help advance fundamental knowledge in sperm physiology involving epigenetic and genetic determinants. The new knowledge can be used to enhance reproductive biotechnology to improve fertility. In addition, the data generated using the unique bull model can be applied to study mammalian reproduction and development due to similarities in genetics and physiology between bovine and other mammals.

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