Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes.

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.

Emotion-attention network interactions during a visual oddball task.

Emotional and attentional functions are known to be distributed along ventral and dorsal networks in the brain, respectively. However, the interactions between these systems remain to be specified. The present study used event-related functional magnetic resonance imaging (fMRI) to investigate how attentional focus can modulate the neural activity elicited by scenes that vary in emotional content. In a visual oddball task, aversive and neutral scenes were presented intermittently among circles and squares. The squares were frequent standard events, whereas the other novel stimulus categories occurred rarely. One experimental group [N=10] was instructed to count the circles, whereas another group [N=12] counted the emotional scenes. A main effect of emotion was found in the amygdala (AMG) and ventral frontotemporal cortices. In these regions, activation was significantly greater for emotional than neutral stimuli but was invariant to attentional focus. A main effect of attentional focus was found in dorsal frontoparietal cortices, whose activity signaled task-relevant target events irrespective of emotional content. The only brain region that was sensitive to both emotion and attentional focus was the anterior cingulate gyrus (ACG). When circles were task-relevant, the ACG responded equally to circle targets and distracting emotional scenes. The ACG response to emotional scenes increased when they were task-relevant, and the response to circles concomitantly decreased. These findings support and extend prominent network theories of emotion-attention interactions that highlight the integrative role played by the anterior cingulate.

Later Life Consequences of Developmental Mitochondrial DNA Damage in C. elegans

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.

Atypical Fibroxanthoma of the Bulbar Conjunctiva.

PURPOSE: To report a rare case of atypical fibroxanthoma (AFX) of the bulbar conjunctiva, and to compare it with previously published cases of conjunctival AFX. METHODS: A 37-year-old woman developed a growth on the bulbar conjunctiva of her left eye that increased in size and redness over 4 months and was associated with blurry vision in the left eye, occasional diplopia, irritation of the eye, and increasing tearing. The mass was surgically excised. RESULTS: Slit-lamp examination disclosed a highly vascularized conjunctival lesion with intact lustrous epithelium and a raised nodular edge encroaching on the nasal corneal limbus of the left eye. Pathological examination and immunohistochemistry were diagnostic of AFX. CONCLUSIONS: AFX of the conjunctiva is rare, with this being only the fifth example of this neoplasm reported at this site. Complete surgical excision is the most appropriate treatment option.