Summary Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1–PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.

Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of -regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping -regulatory elements with the ability to efficiently promote transcription.

Aquaporins (AQPs) are an ubiquitous family of proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs. At present, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), or also glycerol and other small solutes (AQP3, AQP7, AQP9, AQP10, AQP12). The genomic, structural, and functional aspects of this family are briefly described. In particular, proteomic approaches to identify and characterize the most studied AQPs, mainly through SDS-PAGE followed by MS analysis, are discussed. Moreover, the clinical importance of the best studied aquaporin (AQP1) in human diseases is also provided.

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.