DEFINING THE FUNCTIONS OF USP22 AND USP44 IN REGULATION OF H2BUB1 LEVELS

Aberrant levels of histone ubiquitination are involved in various human diseases including neurodegenerative disorders and cancers. Particularly, Histone H2B monoubiquitination (H2Bub1) is highly associated with gene regulation in both normal cells and diseases. Many deubiquitinases (mainly USPs) are defined to regulate global H2Bub1 levels. However, how these USPs are regulated and how they contribute to diseases are not well understood. USP22, part of the deubiquitination module (DUBm) in the SAGA complex, is a well-defined regulator of H2Bub1 levels. ATXN7, another crucial subunit of the SAGA DUBm, is involved in a neurodegenerative disease, spinocerebellar ataxia type 7 (SCA7), due to a polyglutamine (polyQ) expansion in its N-terminal region. Given the allosteric regulation of USP22 DUB activity within the DUBm, whether and how the polyQ expansion in ATXN7 affects SAGA DUB activity was not known. To address this question, we reconstituted the SAGA DUBm in vitro with wild type ATXN7 or the ATXN7polyQ mutant. We found that the DUBm has minimal activity in the absence of ATXN7. ATXN7-92Q, which corresponds to a pathogenic form of the protein, is largely insoluble unless it is incorporated into the DUBm. Importantly, we demonstrated that ATXN7-92Q enhances DUB activity to a similar extent as WT ATXN7. Consistent with these in vitro results, co-overexpression of DUBm components in human astrocytes promotes the solubility of ATXN7-92Q, and greatly inhibits its aggregation into nuclear inclusions that sequester other DUBm components including ATXN7L3, leading to global increases in H2Bub levels. Co-overexpression of ATXN7L3 and ENY2 with ATXN7-92Q reverses this effect. Finally, we found that global H2Bub levels are increased in cerebellums taken from a SCA7 mouse model, consistent with reduced DUB activity in the presence of ATXN7 polyQ mutant. Taken together, our study suggests that ATXN7-polyQ expansions do not change the biochemical activity of the DUBm, but they likely contribute to SCA7 by initiating aggregate formation that sequesters DUBm components away from H2B and other substrates. To further study the contribution of the DUB activity of USP22 to SCA7 disease, we also planned to delete USP22 in Purkinje cells and Bergman glia, both of which are involved in the pathogenesis of SCA7 disease, through establishing a FloxP-Usp22 mouse model. We generated Usp22FloxFRT mice by in vitro fertilization using sperm obtained from the Knock Out Mouse Project (KOMP). After crossing with FLpase mice, we obtained the Usp22Flox mouse model. Currently, we are crossing Usp22Flox mice with Pcp2-Cre and GFAP-Cre mice, which will specifically delete Usp22 in Purkinje cells and Bergman glia, respectively. We will analyze the potential “Ataxia” phenotypes of the mice with Usp22 deletion in Purkinje cells or Bergman glia. Similar to USP22, USP44 is also involved in H2Bub1 regulation. The USP44 deubiquitinase is implicated in various biological processes including ES cell differentiation, DNA damage repair, and cancer development. However, USP44 alone is not active, and whether USP44 directly deubiquitinates histone H2B and what other partners are required for its DUB activity are not known. Using tandem affinity purification and Multidimensional Protein Identification Technology (MudPIT), we identified USP44 as an integral subunit of the Nuclear receptor co-repressor complex (N-CoR complex). As part of N-CoR, USP44 deubiquitinates H2B in vitro and in vivo. Depletion of USP44 impairs the repressive activity of N-CoR complex targeted to a chromatinized luciferase reporter. ChIP-qPCR experiments indicate that USP44 is recruited to the promoter of luciferase reporter, leading to decreased H2Bub levels. These data indicate that USP44 and HDAC3 work together within N-CoR, to repress transcription of target genes. Furthermore, USP44 expression is higher, and H2Bub1 levels are lower, in MDA-MB-231 triple negative breast cancer cells than in MCF-10A cells. Depletion of USP44 impairs invasiveness of MDA-MB-231 cells in vitro and leads to an increase of global H2Bub1 levels, as does depletion of another essential subunit of NCoR, TBL1XR1. Together, our findings indicate that USP44 and N-CoR may contribute to metastatic behaviors of triple negative breast cancer cells. In summary, both USP22 and USP44 participate in multiple biological processes through modulating H2Bub1 levels and defective activity of these DUBs likely contributes to various human diseases. Our data highlight the importance of appropriate DUB activity and H2Bub1 levels for normal cell growth and behavior

Grazing weakens N-addition effects on soil greenhouse gas emissions in a semi-arid grassland

Grazing and anthropogenic nitrogen (N) enrichment co-occur in most grassland ecosystems and may have substantial effects on production of soil greenhouse gases (GHGs). Although the individual effects of N addition and grazing on soil GHGs are well understood, their long-term interactive effects on grassland soil GHGs remain unclear. We conducted seven-year in situ measurement of three major GHGs in a long-term experiment comprising grazing (no, light, moderate, and heavy grazing intensity) and N-addition treatments (control, N addition: 10 g N m−2 year−1) in a semi-arid grassland, to determine the effects of N addition and grazing on GHGs. We found that moderate grazing reduced cumulative CO2 emissions by 10%–11% compared with no, light, and heavy grazing. Unusually, CH4 emissions from soils and N2O uptake were found in this semi-arid grassland. Soil CH4 uptake was markedly inhibited by moderate and heavy grazing. Relative to no grazing, grazing significantly reduced 60%–88% N2O uptake over seven years on average. Nitrogen addition alone increased cumulative CO2 emissions by 16% relative to control. An antagonistic effect between grazing and N addition was found on cumulative CO2 emissions, cumulative CH4 uptake, and global warming potential (GWP). Light grazing on this semi-arid grassland could offset 14% of the soil GHG emissions induced by N addition. Soil NO3 − -N was the most important factor controlling soil CO2 emissions and CH4 uptake, and soil pH was a major factor mediating soil N2O uptake or consumption. Our study highlights the importance that adjusting the grazing intensity of grassland is one of efficient strategies to mitigate GHGs emissions in the context of climate change

USP44 Is an Integral Component of N-CoR that Contributes to Gene Repression by Deubiquitinating Histone H2B

SummaryDecreased expression of the USP44 deubiquitinase has been associated with global increases in H2Bub1 levels during mouse embryonic stem cell (mESC) differentiation. However, whether USP44 directly deubiquitinates histone H2B or how its activity is targeted to chromatin is not known. We identified USP44 as an integral subunit of the nuclear receptor co-repressor (N-CoR) complex. USP44 within N-CoR deubiquitinates H2B in vitro and in vivo, and ablation of USP44 impairs the repressive activity of the N-CoR complex. Chromatin immunoprecipitation (ChIP) experiments confirmed that USP44 recruitment reduces H2Bub1 levels at N-CoR target loci. Furthermore, high expression of USP44 correlates with reduced levels of H2Bub1 in the breast cancer cell line MDA-MB-231. Depletion of either USP44 or TBL1XR1 impairs the invasiveness of MDA-MB-231 cells in vitro and causes an increase of global H2Bub1 levels. Our findings indicate that USP44 contributes to N-CoR functions in regulating gene expression and is required for efficient invasiveness of triple-negative breast cancer cells