CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy) 2 (dpqp)] 2+ ) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule.[Ru(bpy) 2 (dpqp)] 2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-β (Aβ) oligomers increase. Our assays show that Aβ forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS−PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aβ oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different times.

In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These fi ndings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis.

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA-DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G-C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.