Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell-type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways are not yet understood. Herein, we report the cellular responses to peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered the accumulation of its substrate TYSND1 protease, while the overall expression of peroxisomal proteins, as well as TYSND1-dependent ACOX1 processing appeared normal, reflecting early stages of peroxisomal proteotoxic stress. Consequently, the alteration of peroxisome size and numbers, and luminal protein import failure was coupled with induction of cell-specific cellular stress responses. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight into our understanding of the tissue-specific responses seen in peroxisomal disorders.

Profiling the Epigenetic Landscape of the Tumor Microenvironment Using Chromatin Immunoprecipitation Sequencing.

Cancer cells within a tumor exhibit phenotypic plasticity that allows adaptation and survival in hostile tumor microenvironments. Reprogramming of epigenetic landscapes can support tumor progression within a specific microenvironment by influencing chromatin accessibility and modulating cell identity. The profiling of epigenetic landscapes within various tumor cell populations has significantly improved our understanding of tumor progression and plasticity. This protocol describes an integrated approach using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) optimized to profile genome-wide post-translational modifications of histone tails in tumors. Essential tools amenable to ChIP-seq to isolate tumor cell populations of interest from the tumor microenvironment are also presented to provide a comprehensive approach to perform heterogeneous epigenetic landscape profiling of the tumor microenvironment.

A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia.

In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.

Epistasis, not numbers, regulates functions of clustered Dahl rat quantitative trait loci applicable to human hypertension.

Quantitative trait loci (QTLs) for blood pressure (BP) were found on chromosome 10 of Dahl salt-sensitive rats and are potentially important to human essential hypertension. But their identities and how they influence BP together were not known. Presently, we first fine mapped existing QTLs, C10QTL1, C10QTL2, and C10QTL3, by constructing congenic strains. In the process, a new QTL, C10QTL4, was identified. Because the intervals harboring C10QTL1 and C10QTL4 contain a maximum of 16 and 10 possible genes, respectively, a limited number of specific gene targets has been identified to be QTLs residing in human homologous regions on chromosome 17. Moreover, because none of these candidates encodes a gene known to influence BP, the 2 QTLs will represent novel genes for BP regulations. Second, we used congenic strains with QTL combinations to analyze the interactions between the QTLs. Consequently, a double combination of C10QTL4 and C10QTL1 possessed the same BP as each of the 2 QTLs alone. BP of a triple combination of C10QTL4, C10QTL1, and C10QTL3 was not different from BP of the C10QTL4 and C10QTL1 double combination. These results demonstrate that C10QTL4, C10QTL1, and C10QTL3 are epistatic to one another in their BP effects. In contrast, when adding C10QTL2 into the triple formation of the 3 QTLs above to create a quadruple QTL combination, BP increased proportionately, indicating that C10QTL2 acts independently of C10QTL4, C10QTL1, and C10QTL3. The epistatic and additive interactions uncovered in the animal model will help elucidate similar interactions playing a role in human essential hypertension.

A loss of genome buffering capacity of Dahl salt-sensitive model to modulate blood pressure as a cause of hypertension.

Essential hypertension is a complex trait influenced by multiple genes known as quantitative trait loci (QTLs) for blood pressure (BP). It is not clear, however, what roles these QTLs play in maintaining normotension. Insights gained toward the maintenance of normotension will shed light on how hypertension can result from a deficiency or malfunctioning of this maintenance. Currently, congenic strains were systematically constructed using Dahl salt-sensitive (DSS) and Lewis (LEW) rats not only to define QTLs (i.e. in DSS background), but also to ascertain effects of the same QTLs in preserving normotension (i.e. in LEW background), a first such study. Results showed that although LEW alleles for two QTLs on Chromosome (Chr) 18 lowered BP on the DSS background, their BP-increasing DSS alleles failed to influence BP in the LEW background. To further prove that the LEW background is resistant and the DSS background is susceptible to the effects of QTLs, BP-increasing alleles of a QTL on Chr 2 were introgressed into the DSS background, and its BP-decreasing alleles into the LEW background. Indeed, there was no BP-decreasing effect on the LEW background while demonstrating a BP-increasing effect on the DSS background. Thus, a genetic regulation of BP QTLs in the LEW genome inhibits BP changes by nullifying the effects of BP-altering QTLs. In comparison, the DSS genome must have lost the buffering capacity for stabilizing BP. The current work presents good evidence that a lack of regulation for functions of BP QTLs is a potential underlying cause of hypertension.

Complete and overlapping congenics proving the existence of a quantitative trait locus for blood pressure on Dahl rat chromosome 17.

Linkage studies suggested that a quantitative trait locus (QTL) for blood pressure (BP) was present in a region on chromosome 17 (Chr 17) of Dahl salt-sensitive (DSS) rats. A subsequent congenic strain targeting this QTL, however, could not confirm it. These conflicting results called into question the validity of localization of a QTL by linkage followed by the use of a congenic strain made with an incomplete chromosome coverage. To resolve this issue, we constructed five new congenic strains, designated C17S.L1 to C17S.L5, that completely spanned the +/-2 LOD confidence interval supposedly containing the QTL. Each congenic strain was made by replacing a segment of the DSS rat by that of the normotensive Lewis (LEW) rat. The only section to be LL homozygous is the region on Chr 17 specified in a congenic strain, as evidenced by a total genome scan. The results showed that BPs of C17S.L1 and C17S.L2 were lower (P < 0.04) than that of DSS rats. In contrast, BPs of C17S.L3, C17S.L4, and C17S.L5 were not different (P > 0.6) from that of DSS rats. Consequently, a BP QTL must be located in an interval of approximately 15 cM shared between C17S.L1 and C17S.L2 and unique to them both, as opposed to C17S.L3, C17S.L4, and C17S.L5. The present study illustrates the importance of thorough chromosome coverage, the necessity for a genome-wide screening, and the use of "negative" controls in physically mapping a QTL by congenic strains.

Combining congenic coverage with gene profiling in search of candidates for blood pressure quantitative trait loci in Dahl rats.

Chromosomes (Chr) 10 and 16 of the Dahl salt-sensitive (S) rat harbor quantitative trait loci (QTLs) for blood pressure (BP). To facilitate gene discovery of these QTLs, gene profiling based on microarrays was combined with fine QTL mapping to identify potential candidate genes that are differentially expressed. First, the region harboring the BP QTL on Chr 16 was narrowed by comparative congenic mapping. In this endeavor, a number of new chromosome markers were generated and used to physically define the chromosome interval in question. Second, in an effort to minimize the costs of gene profiling without sacrificing the chance of gene discovery, a combination congenic strain was produced by replacing one segment of Chr 10 along with one segment of Chr 16 of the hypertensive S rat by those of the normotensive Lewis (LEW) rat. Both of these regions are known to contain BP QTLs. Third, kidneys of this combination congenic strain and the S strain were employed for expression profiling studies. Finally, a comparison between the two strains yielded a number of potentially differentially expressed candidates. Six Established Sequence Tags (ESTs)/genes among them were located in Chr 10 regions and 1 was found in a Chr 16 region, and the genetic make-ups of all these regions were shown to be different between S and LEW. However, none of these ESTs/genes identified by gene profiling were located in an interval containing a QTL. Thus, the present study highlights the importance of correlating the results of gene expression profiling with fine congenic mapping.

Comprehensive congenic coverage revealing multiple blood pressure quantitative trait loci on Dahl rat chromosome 10.

Chromosome mapping based on congenic strains can restrict quantitative trait loci (QTLs) for blood pressure (BP) into small intervals that are otherwise indistinguishable in linkage analysis. Also, congenic strains can be created to test a candidate gene to be a BP QTL. Taking full advantage of these features, we produced 10 congenic strains by replacing various segments of chromosome (Chr) 10 of the Dahl salt-sensitive (DSS) rat with those of the Lewis (LEW) rat. These strains were made to systematically cover an entire section of Chr 10. Three of the strains were designed to narrow the intervals that harbor previously mapped QTL1 and QTL2. Two of the strains were designed for the express purpose of testing the QTL candidacy of loci for inducible nitric oxide synthase (Nos2) and angiotensin-converting enzyme (Ace) genes. BPs of these strains were measured by telemetry and compared with those of the DSS rat. Consequently, QTL1 and QTL2 were narrowed to segments of 53.5 and 100.4 centiRays, respectively. A new QTL, QTL3, was found between QTL1 and QTL2. Both Nos2 and Ace have been disqualified as QTLs in the DSS and LEW comparison. Therefore, there are no obvious candidate genes in the segments that harbor these 3 QTLs, which represent genes previously not thought to be involved in BP regulation. These QTLs will likely have an influence on studies of human hypertension because of their homology with the human CHR 17 region in which QTLs for BP have been found.