The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Investigation of Scavenger Receptor Class B Type I gene variants in patients with coronary heart disease with a history of early myocardial infarction.

OBJECTIVE: The scavenger receptor class B type 1 (SR-BI, SCARB1), which is a high-density lipoprotein (HDL) receptor that mediates selective cholesteryl ester uptake, plays an important role in reverse cholesterol transport. This study investigated the distribution of polymorphic variants of the SR-BI gene in patients with coronary heart disease (CHD) with a history of early myocardial infarction (MI) at an early age and their effects on their serum lipid levels. METHODS: SR-BI rs5888(T>C), rs4238001(C>T), and rs10846744(G>C) were analyzed in 100 male patients with CHD with a history of MI (MI+) who were younger than 50 years and 89 male control subjects without MI history (MI-) using real-time polymerase chain reaction (PCR) and mutant-allele-specific PCR techniques. RESULTS: SR-BI rs4238001 common-CC genotype was found to be more frequent in patients with MI+ than in control subjects (MI-; odds ratio 4.046, p<0.001). The rs10846744 rare-C allele showed a significant association with increased total cholesterol (p=0.014) and triglyceride (p=0.009) levels in the MI+ CHD group. Logistic regression analysis confirmed that there may be an association between the rs4238001-CC genotype (p=0.002), smoking (p=0.026), and MI+ CHD in the presence of other risk factors associated with CHD, whereas haplotype analysis confirmed that patients with MI+ CHD (rs5888-C, rs10846744-G, and rs4238001-C alleles) and CCC (rs5888-C, rs10846744-C, and rs4238001-C alleles) haplotypes were highly frequent (p<0.01 and p=0.027, respectively). CONCLUSION: These results indicated that SR-BI gene variants show different distribution in patients with MI+ CHD compared with that in MI- control subjects, and these variants may have effects in favor of dyslipidemia.

Long Term Rescue of the TSH Receptor Knock-Out Mouse - Thyroid Stem Cell Transplantation Restores Thyroid Function.

The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500µU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.