Novel Telomere-Anchored PCR Approach for Studying Sexual Stage Telomeres in Aspergillus nidulans

Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed telomere-anchored PCR, measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms

Genome Wide DNA Copy Number Analysis of Serous Type Ovarian Carcinomas Identifies Genetic Markers Predictive of Clinical Outcome

Ovarian cancer is the fifth leading cause of cancer death in women. Ovarian cancers display a high degree of complex genetic alterations involving many oncogenes and tumor suppressor genes. Analysis of the association between genetic alterations and clinical endpoints such as survival will lead to improved patient management via genetic stratification of patients into clinically relevant subgroups. In this study, we aim to define subgroups of high-grade serous ovarian carcinomas that differ with respect to prognosis and overall survival. Genome-wide DNA copy number alterations (CNAs) were measured in 72 clinically annotated, high-grade serous tumors using high-resolution oligonucleotide arrays. Two clinically annotated, independent cohorts were used for validation. Unsupervised hierarchical clustering of copy number data derived from the 72 patient cohort resulted in two clusters with significant difference in progression free survival (PFS) and a marginal difference in overall survival (OS). GISTIC analysis of the two clusters identified altered regions unique to each cluster. Supervised clustering of two independent large cohorts of high-grade serous tumors using the classification scheme derived from the two initial clusters validated our results and identified 8 genomic regions that are distinctly different among the subgroups. These 8 regions map to 8p21.3, 8p23.2, 12p12.1, 17p11.2, 17p12, 19q12, 20q11.21 and 20q13.12; and harbor potential oncogenes and tumor suppressor genes that are likely to be involved in the pathogenesis of ovarian carcinoma. We have identified a set of genetic alterations that could be used for stratification of high-grade serous tumors into clinically relevant treatment subgroups

Dynamic enhancers control skeletal muscle identity and reprogramming.

Skeletal muscles consist of fibers of differing metabolic activities and contractility, which become remodeled in response to chronic exercise, but the epigenomic basis for muscle identity and adaptation remains poorly understood. Here, we used chromatin immunoprecipitation sequencing of dimethylated histone 3 lysine 4 and acetylated histone 3 lysine 27 as well as transposase-accessible chromatin profiling to dissect cis-regulatory networks across muscle groups. We demonstrate that in vivo enhancers specify muscles in accordance with myofiber composition, show little resemblance to cultured myotube enhancers, and identify glycolytic and oxidative muscle-specific regulators. Moreover, we find that voluntary wheel running and muscle-specific peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) transgenic (mTg) overexpression, which stimulate endurance performance in mice, result in markedly different changes to the epigenome. Exercise predominantly leads to enhancer hypoacetylation, whereas mTg causes hyperacetylation at different sites. Integrative analysis of regulatory regions and gene expression revealed that exercise and mTg are each associated with myocyte enhancer factor (MEF) 2 and estrogen-related receptor (ERR) signaling and transcription of genes promoting oxidative metabolism. However, exercise was additionally associated with regulation by retinoid X receptor (RXR), jun proto-oncogene (JUN), sine oculis homeobox factor (SIX), and other factors. Overall, our work defines the unique enhancer repertoires of skeletal muscles in vivo and reveals that divergent exercise-induced or PGC1α-driven epigenomic programs direct partially convergent transcriptional networks

A Stringent Systems Approach Uncovers Gene-Specific Mechanisms Regulating Inflammation

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks

Loss of Transcriptional Repression by BCL6 Confers Insulin Sensitivity in the Setting of Obesity

Summary: Accumulation of visceral adiposity is directly linked to the morbidity of obesity, while subcutaneous body fat is considered more benign. We have identified an unexpected role for B cell lymphoma 6 (BCL6), a critical regulator of immunity, in the developmental expansion of subcutaneous adipose tissue. In adipocyte-specific knockout mice (Bcl6AKO), we found that Bcl6 deletion results in strikingly increased inguinal, but not perigonadal, adipocyte size and tissue mass in addition to marked insulin sensitivity. Genome-wide RNA expression and DNA binding analyses revealed that BCL6 controls gene networks involved in cell growth and fatty acid biosynthesis. Using deuterium label incorporation and comprehensive adipokine and lipid profiling, we discovered that ablation of adipocyte Bcl6 enhances subcutaneous adipocyte lipogenesis, increases levels of adiponectin and fatty acid esters of hydroxy fatty acids (FAHFAs), and prevents steatosis. Thus, our studies identify BCL6 as a negative regulator of subcutaneous adipose tissue expansion and metabolic health. : Senagolage et al. identify BCL6 as a key regulator of body fat distribution. BCL6 directly represses fatty acid biosynthetic and growth genes in adipocytes. Mice constitutively lacking adipocyte Bcl6 exhibit expansion of their subcutaneous adipose tissue, enhanced insulin sensitivity, and protection from hepatic steatosis. Keywords: obesity, BCL6, BCL-6, repressor, adipocyte, hypertrophy, subcutaneous fat, insulin sensitivity, lipogenesis, adipose tissue, FAHFA, fatty acid esters of hydroxy fatty acids, PAHSA, adiponectin, ceramid

Alignment of an <i>A. nidulans</i> ORF to known TERT motifs.

<p>The ANID_03753.1 predicted protein sequence (AN_TERT) was aligned to the TERT sequence from mouse (MOUSE_TERT, accession number O70372) and <i>S. pombe</i> (SP_TERT, accession number O13339) using the Align program in Vector NTI (Invitrogen). The region of the alignment including AN_TERT residues 601 through 1120 were selected and are shown. The conserved telomerase reverse transcriptase motifs (Motif T, 1, 2, Motif A, Motif B, Motif C, Motif D and Motif E) are indicated above the aligned sequences. Residues identical in all three sequences are in shown with dark shading, whereas sequences that are similar or identical in only two sequences are shown in lighter shading.</p

Telomere-anchored PCR assay detects telomeres in <i>A. nidulans.</i>

<p><b>A.</b> The design of telomere-anchored PCR of C-tailed DNA using chromosome II-L. Forward primers were constructed at three different positions, labeled A, B, and C. Reverse primers were made to either a “G-only” sequence or to a G sequence that contained all six possible terminal sequences at the 3′end. <b>B.</b> PCR yielded products of expected sizes when telomere-anchored PCR primer 1 or 4 (TAP) were used with primer C (lanes 2 and 3, respectively) or primer B (lanes 4 and 5, respectively), and when telomere-anchored PCR primer 4 was used with primer A (lane 8) or primer B (lane 9). No PCR product was detected on non-tailed template without terminal transferase (TdT), using telomere-anchored PCR primer 4 and primer A (lane 11) or primer B (lane 12). The positive controls in lanes 6, 10, and 13 used primer A and a reverse primer internal to the telomere.</p

All permutations are present at the ultimate telomeric repeat.

<p><b>A.</b> PCR products were obtained for all six telomere anchored PCR primers with no significant variation in intensity, indicating all six permutations at the ultimate sequence were amplified equally. The positive control used primers E and F. The template DNA was from an <i>nku-</i> strain. <b>B.</b> DNA sequence analysis performed on the PCR products of telomere-anchored primers 1 and 4 from panel A showed the presence of a G tract (highlighted in yellow) and the first telomere repeat (bracketed in red) as expected. The first synthesized telomeric repeat is shown in the second red bracket. The number of Gs varied from one cloned sequence to the next, and is indicated as “Gs” to the left. The number of telomeric repeats is shown at right. <b>C.</b> The template DNA was from strain GR5 having a wild-type <i>nkuA</i> gene. The telomere-anchored PCR was run using primer A coupled with the six different telomere-anchored permutation primers. PCR products were seen for all the telomere-anchored primers.</p

Telomerase mutants showed dramatic shortening of telomeres compared to wild-type as assayed by telomere-anchored PCR.

<p>A. Genomic DNA was isolated from wild-type strains (GR5 in lane 1, TN02A7 in lane 2) and two putative <i>trtA</i> deletion mutants (TDP3-2 in lane 3, TDP3-32 in lane 4), digested with EcoRI and analyzed by Southern blot using PCR product A as probe. B. Telomere-anchored PCR was performed on genomic DNA isolated from wild-type and <i>trtA</i> deletion mutants with either forward primer A or B coupled with telomere-anchored PCR primer 4. At both eight hours and sixteen hours after transformation, <i>trtA</i> deletion mutants show significantly shorter telomeres (lanes 7-10, red dot in lane 8) than wild-type (lanes 1-2) and <i>nkuA</i> deletion mutant (lanes 3-6, blue dot in lane 4).</p