H1t is a novel linker histone variant synthesized in mid- to late pachytene spermatocytes. Its regulatory region is of interest because developmentally specific expression has been impressed on an otherwise ubiquitously expressed promoter. Using competitive band-shift assays and specific antisera, we have now shown that the H1t-60 CCTAGG palindrome motif region binds members of the RFX family of transcriptional regulators. The testis-specific binding complex contains RFX2, probably as a homodimer. Other DNA-protein complexes obtained from testis as well as somatic organs contain RFX1, primarily as a heterodimer. Western blots confirmed that RFX2 expression is greatly enhanced in adult testis and that RFX2 is equally prominent in highly enriched populations of late pachytene spermatocytes and round spermatids. Immunohistochemistry carried out on mouse testis showed that RFX2 is strongly expressed in pachytene spermatocytes, remains high in early round spermatids, and declines only in advance of nuclear condensation. Maximum expression correlates well with the appearance of H1t. In contrast, RFX1 immunoreactivity in germ cells was only detected in late round spermatids. RFX-specific band complexes were also identified for both the mouse lamin C2 and Sgy promoters, using either testis nuclear extracts or in vitro-synthesized RFX2. These results call attention to RFX2 as a transcription factor with obvious potential for the regulation of gene expression during meiosis and the early development of spermatids.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. While knockout of Nrf2 exaggerates cardiac pathological remodeling and dysfunction in diverse pathological settings, pharmacological activation of Nrf2 protects against cardiomyocyte injury and cardiac dysfunction. In contrast, there is also a concern that the chronic activation of Nrf2 secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. However, a direct link between cardiac specific activation of Nrf2 and cardiac protection or dysfunction in vivo remains to be established. Therefore, we investigated the effect of cardiomyocyte-specific transgenic activation of Nrf2 (Nrf2ctg) on cardiac pathological remodeling and dysfunction. We found that the cardiomyocyte-specific activation of Nrf2 suppressed myocardial oxidative stress as well as cardiac apoptosis, fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice. Notably, the constitutive activation of Nrf2 increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC. Nrf2 gene gain- and loss-of-function approaches revealed that Nrf2 enhances autophagosome formation and autophagic flux in cardiomyocytes. Unexpectedly, while Nrf2 minimally regulated apoptosis, it suppressed significantly the proteotoxic necrosis in cardiomyocytes. In addition, Nrf2 attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes. Taken together, we demonstrated for the first time that cardiac specific activation of Nrf2 suppresses cardiac maladaptive remodeling and dysfunction most likely by enhancing autophagic clearance of toxic protein aggregates in the heart.

H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths.

Viruses are monodispersed biomacromolecules with well-defined 3-D structures at the nanometer level. The relative ease to manipulate viral coat protein gene to display numerous functional groups affords an attractive feature for these nanomaterials, and the inability of plant viruses to infect mammalian hosts poses little or no cytotoxic concerns. As such, these nanosized molecular tools serve as powerful templates for many pharmacological applications ranging as multifunctional theranostic agents with tissue targeting motifs and imaging agents, potent vaccine scaffolds to induce cellular immunity and for probing cellular functions as synthetic biomaterials. The results herein show that combination of serum-free, chemically defined media with genetically modified plant virus induces rapid onset of key bone differentiation markers for bone marrow derived mesenchymal stem cells within two days. The xeno-free culture is often a key step toward development of ex vivo implants, and the early onset of osteocalcin, BMP-2 and calcium sequestration are some of the key molecular markers in the progression toward bone formation. The results herein will provide some key insights to engineering functional materials for rapid bone repair.