Transcription factors

Cardiovascular disease is a broad term that encompasses a variety of disorders in which the heart and its associated blood vessels lose the capacity to deliver blood efficiently and effectively throughout the body. Cardiac endothelial cells play a vital role in maintaining the homeostatic balance of cardiac physiology. Research into c-Myc, a master regulator involved in the transcription of a large set of genes that regulate inflammation, has been the focus of new therapeutics aimed at treating or lessening the deleterious effects of cardiovascular disease. This project serves to explore how endothelial loss of c-Myc impacts cardiac function under normal and stress conditions, using ultrasound echocardiography image analysis to determine the key differences between all models.

Serine/Arginine splicing factor 1 (SRSF1) is an RNA-binding protein (RBP) with multiple functions in RNA biogenesis. SRSF1 plays a prominent role in oncogenesis, immune function, and response to several physiological stimuli. To date, the role of SRSF1 as a regulator of mRNA splicing has been largely considered the main mechanism driving its biological functions and its role in disease. We have now characterized SRSF1’s role in Human Immunodeficiency Virus Type I (HIV-1) transcription. SRSF1 interacts with the 7SK small nuclear ribonucleoprotein (snRNP) to mobilize and activate the positive transcription-elongation factor (P-TEFb), which is then positioned on the HIV-1 promoter to increase the processivity of RNA polymerase II (RNAPolII) and promote the release of the negative regulators of transcription DSIF/NELF. Next, we defined the role of SRSF1 in the transcription of cellular genes utilizing an RNA sequencing (RNASeq) time course approach was used to detect changes in the transcriptome in response to SRSF1 overexpression. RNASeq data analysis revealed a subset of genes that were upregulated in response to SRSF1 overexpression. Nuclear run-on and qPCR assays experimentally validated 28 of these genes.

L1-type cell adhesion molecule (L1CAM) plays an essential role in the
development of nervous system and is also highly relevant for the progression of diseases
such as Alzheimer’s disease, stroke and cancers, some of the leading causes of human
mortality. In addition to its canonical role as a plasma membrane protein organizing the
cytoskeleton, recent in vitro studies have revealed that transmembrane as well as cytosolic
fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus and
regulate expression of genes involved in DNA post-replication repair, cell cycle control,
migration and differentiation. However, little is known about the in vivo function of
L1CAM in the adult nervous system.
This dissertation research focuses on studying in vivo nuclear translocation and
function of L1CAM. Using the Drosophila model system, we first show that the sole
Drosophila L1CAM homolog, Neuroglian (Nrg), is proteolytically cleaved by Alzheimer’s
associated secretases, similar to L1CAM, and is also translocated to the nucleus in the adult nervous system. Subsequently, we have shown that the deletion of highly conserved
Ankyrin binding domain or FIGQY motif disrupts nuclear import. Further experiments
have revealed that the nuclear translocation of Nrg is in fact regulated by the
phosphorylation of the FIGQY motif. Importantly, our studies also show transgenic
expression of full-length Nrg or the intracellular domain of Nrg resulted in increased myc
expression, which is associated with increased sensitivity to oxidative stress and reduced
life span. On the other hand, deletion of the FIGQY motif or mutations preventing its
phosphorylation led to decrease in myc expression. In summary, we have identified a novel
role for the highly conserved Ankyrin binding domain in nuclear translocation and
transcriptional regulation of the Drosophila myc oncogene, which is of high relevance to
neurodegenerative diseases and cancer associated with oxidative stress.

The classic guidance molecules, Netrin and its receptor Frazzled (Fra), dictate the strength of
synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating
gap junction localization in the pre-synaptic terminal. In Netrin mutant animals the synaptic
coupling between a giant interneuron and the jump motor neuron was weakened. Dye-coupling
between these two neurons was severely compromised or absent. These mutants exhibited
anatomically and physiologically defective synapses between the giant fiber (GF) and
tergotrochanteral motor neuron (TTMn). In cases where Netrin mutants displayed apparently
normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses.
Dye-coupling between the giant fiber and the motor neuron was reduced or eliminated,
suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap
junctions with antibodies to Shaking-B Innexin (ShakB), they were significantly decreased or
absent in the pre-synaptic terminal of the mutant GF. This data is the first to show that Netrin and
Frazzled regulate placement of gap junctions pre-synaptically at a central synapse. In the Drosophila Giant Fiber System, we demonstrate a mechanism that ensures the monoinnervation of two homologous motor neurons by two homologous interneurons. In a scenario where both interneurons could synapse with both motor neuron targets, each interneuron exclusively synapsed with only one target and the circuit functions at normal physiological levels. This innervation pattern depended on the ratio of netrin-to-frazzled expression. When Netrin was over expressed in the system, shifting the ratio in favor of Netrin,
both interneurons synapsed with both target motor neurons and physiological function was reduced. This resulted in the polyinnervationof a single target. In contrast, when Frazzled was over expressed in the system, one interneuron innervated both targets and excluded the remaining interneuron from making any synaptic contact. This resulted in a single interneuron mono-innervating both motor neurons and physiological function was mutant. The orphaned interneuron made no synaptic contact with either motor neuron target. Physiological function was only normal when the Netrin-Frazzled ratio was at endogenous levels and each GF monoinnervated one motor neuron. When we examined the gap junctions at this synapse in experimental animals, there was a significant reduction of gap junction hemichannels in the presynaptic terminal of axons that deviated from normal innervation patterns. While the synapse dyecoupled, the reduction in gap junction hemichannels reduced function in the circuit.

The zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S-transferase (GST)-ZAD domain chimeric proteins for use in pull down protein binding assays,and GST-Zinc finger (ZnF) array domain chimera for electrophoretic mobility shift assays (EMSA). Protein binding assays indicated two putative conserved interactors, similar to the analogous KRAB system in mammals. ... Competitive bindings were carried out to show a specificity of binding conferred by the identified conserved positions. While the consensus binding sites show relatively few similarities, the predicted target genes identified by the consensus binding sites show significant overlap. The nature of this overlap conforms to the known characteristics of the ZAD family but points to a more positive selection to maintain conservation of function.

The assembly and maintenance of central synapses is a complex process, requiring myriad genes and their products. Highwire is a large gene containing a RING domain, characteristic of ubiquitin E3 ligases. Highwire has been shown to restrain axon growth and control synaptogenesis at a peripheral synapse. Here I examine the roles of Highwire at a central synapse in the adult Drosophila Giant Fiber System (GFS). Highwire is indeed necessary for proper axonal growth as well as synaptic transmission in the GFS. Differences arise between the central synapse and the neuromuscular junction (NMJ), where highwire was initially characterized : expresion from the postsynaptic cell can rescue highwire synaptic defects, which is not seen at the NMJ. In addition, a MAP kinase signaling pathway regulated by highwire at the NMJ has differing roles at a central synapse. Wallenda MAPK can rescue not only the highwire anatomical phenotype but also the defects seen in transmission. Another distinction is seen here : loss of function basket and Dfos enhance the highwire anatomical phenotype while expression of dominant negative basket and Dfos suppress the highwire phenotype. As a result we have compared the signaling pathway in flies and worms and found that the NMJ in the two organisms use a parallel pathway while the central synapse uses a distinct pathway.

Transcriptional regulation of genes is vital to cell success making it an important aspect of research. Transcriptional regulation can occur in many ways; transcription factors bind to the promoter region and block transcription, disrupt an activator protein, or interact with histones to lead to higher order chromatin. Plant HomeoDomain can recognize and bind to different methylation states of histone tails. PHD proteins use other functional regions to carry out functions. Two associated domains having DNA-binding capacity were characterized in this study; the ARID domains of JARID1A and JARID1C and the DDT domains of BAZ1A, BAZ1B and BAZ2A. These genes are important because of their roles in various diseases such as cancer. The consensus sequences for BAZ1A-DDT is GGACGGRnnGG, GnGAGRGCRnnGGnG, RAGGGGGRnG and CRYCGGT. Consensus sequences for BAZ1B-DDT were CGnCCAnCTTnTGGG and YGCCCCTCCCCnR. Consensus sequences for BAZ2A-DDT were TACnnAGCnY and CnnCCRGCnRTGnYY. Consensus sequence for JARID1A-ARID was GnYnGCGYRCYnCnG. Consensus sequences for JARID1C-ARID was RGGRGCCRGGY.

Longitudinals lacking gene (LOLA) is a transcription factor that is involved in a variety of axon guidance decisions in Drosophila melanogaster nervous system. Besides having a role as an epigenetic silencer and in the programmed cell death in Drosophila's ovary, this gene is also an example of complex transcription unit. LOLA is a transcription repressor and can generate 17 DNA - binding isoforms, through alternative splicing, each containing distinct zinc-finger proteins. This unique DNAbinding binding sequence to which LOLA-ZFP binds has been determined for four of the lola isoforms F, J, P and K. Also, bioinformatics' tool approach has been taken to identify the target genes that are regulated by these four LOLA splice variants. Future work will be done for the five other LOLA isoforms to categorize their putative DNA-binding sequences and subsequently their protein interactions.

Transcription factors are macromolecules that are involved in transcriptional regulation by interacting with specific DNA regions, and they can cause activation or silencing of their target genes. Gene regulation by transcriptional control explains different biological processes such as development, function, and disease. Even though transcriptional control has been of great interest for molecular biology, much still remains unknown. This study was designed to generate the most current list of human transcription factor genes. Unique entries of transcription factor genes were collected and entered into Microsoft Office 2007 Access Database along with information about each gene. Microsoft Office 2007 Access tools were used to analyze and group collected entries according to different properties such as activator or repressor record, or presence of certain protein domains. Furthermore, protein sequence alignments of members of different groups were performed, and phylogenetic trees were used to analyze relationship between different members of each group. This work contributes to the existing knowledge of transcriptional regulation in humans.

The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins.