Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

This study links a noncoding DNA variant to long range regulation of IL-32 isoform expression, modulating susceptibility to HIV.

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.Keywords: Chromatin; Nuclear lamina; Lamin; Nuclear pore complex; Nucleoporin; Nup Chromatin organization by compactionTo fit into the limited space of the nucleus and still carry out its function, human genomic DNA is extensively folded, making it about 10 000-fold more compact. Several levels of compaction have been described: the nucleosome, the 30 nm fiber and higher order chromatin structure.The lowest level of chromatin compaction is the nucleosome. A 5-10-fold compaction is achieved when 146-165 base pairs of DNA are wound around an octamer of histone proteins, which is referred to as the nucleosome core particle. Besides providing a structural basis for the first compaction level, histones can also affect chromatin organization by being chemically modified at their tail or by being replaced by variants of the core histones. These modifications have a major impact on chromatin structure and gene expression by influencing the binding of proteins to the nucleosome, the affinity of DNA for the histone octamer and the stability of higher order structures [1]. Thus, at this low level of organization the nucleosome offers a powerful mechanism for controlling chromatin structure in a local, non-random manner.Findings on the second level of compaction are more ambiguous. In vitro, oligonucleosomes are able to organize themselves into a compact fiber with a diameter of 30 nm in absence of nuclear proteins but in the presence of divalent cations. In vivo, estimated nuclear cation concentrations are even higher than the concentration used in experiments, aiding the compaction [2]. This compaction could be further modulated by the involvement of numerous nuclear proteins in vivo. For example, histone tails and histone H1 further stabilize this structure by binding to linker DNA.All condensation levels above the 30 nm fiber are indicated as higher order chromatin structure. This poorly defined structure may consist of several levels of condensation and is very dynamic and thus hard to study. The question has even been raised whether there is a uniform higher order structure at all, or whether chromatin is too dynamic to form stable structures at a higher order level [3].All levels of compaction are not equal throughout the cell, leading to more accessible and less accessible regions. D...

A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.