Jessica Sarthi

Stanford University | SU · Department of Pediatrics

Histone acetylation of chromatin plays a key role in promoting the dynamic transcriptional responses in neurons that influence the neuroplasticity linked to cognitive ability, yet the specific histone acetyltransferases (HATs) that create such epigenetic marks remain to be elucidated. Here we use the Drosophila neuromuscular junction (NMJ) as a wel...

Axonal transport defects and axonopathy are prominent in early preclinical stages of Alzheimer's disease (AD), often preceding known disease-related pathology by over a year. As epigenetic transcriptional regulatory mechanisms, such as histone acetylation, are critical for neurogenesis, it is postulated that their misregulation might be linked to e...

Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic pl...

Tip60 is a key histone acetyltransferase (HAT) enzyme that plays a central role in diverse biological processes critical for general cell function; however, the chromatin-mediated cell-type specific developmental pathways that are dependent exclusively upon the HAT activity of Tip60 remain to be explored. Here, we investigate the role of Tip60 HAT...

Cognitive decline is a debilitating hallmark during preclinical stages of Alzheimer’s disease (AD), yet the causes remain unclear. Because histone acetylation homeostasis is critical for mediating epigenetic gene control throughout neuronal development, we postulated that its misregulation contributes to cognitive impairment preceding AD pathology....

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localizati...

Summary of mutant phenotypes. (PDF)

Sir4 Sumoylation in mutant backgrounds. Sumoylated proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously [48, 57, 80, 85] followed by western blotting with αMyc antibodies to visualize sumoylated proteins in cells containing Myc-tagged Sir4 with un-tagged Smt3 wild type (JC3433), or His8-tagged Smt3 in wild type...

Strains used in this study. (PDF)

TERRA expression and telomeres length in smc6-9 mutants. (A and B) TERRA expression was determined by RT-qPCR for Tel1R and Tel6R, X only telomeres, at 28C (A) and 34C (B). Statistical significance with p values < .05 (*) or < .01(**) are reported from a two-tailed t-test. (C) Telomere length was determined as in Fig 1F by Southern blot analysis on...

qPCR primers used in this study. (PDF)

ChIP performed on Rap1Myc and Rif1Myc and in non-tagged (nt) strains. ChIP was perform with Chromatin immunoprecipitation (ChIP) was performed on (A) Rap1Myc in wild type (JC2381) and nse3-1 (JC3272), (B) Rif1Myc in wild type (JC3277) and nse3-1 (JC3295), (C) α Myc in non-tagged wild type (JC470) and nse3-1 (JC3607) cells and (D) α FLAG in non-tagg...

Rif1, Rif2 and Smc6 recruitment at native telomeres in various mutant cells. (A) Yeast-two Hybrid analysis was performed as previously described [48]. NSE3 full-length, nse3(1–150)—N-terminal end, nse3(150–300)—C-terminal end, or the nse3-1 mutant were cloned into bait plasmid (pEG202) and RIF2 into prey plasmid (pJG4-6) [86]. Plasmids containing b...

TERRA expression levels in rif2Δ and nse3-1 mutants. (A and B) TERRA expression was determined for Y’ at 28°C and 34°C in wild type (JC470), nse3-1 (JC3607), rif2Δ (JC2992), nse3-1 rif2Δ (JC3269), sir4Δ (JC3737), nse3-1 sir4Δ (JC3741), and sir4Δ rif2Δ (JC3738). Statistical significance with p values < .05 (*) or < .01 (**) are reported from a two-t...

ChIP of yKu70 at telomeres in nse3-1 mutant and wild type cells. Chromatin immunoprecipitation (ChIP) was performed on yKu70Myc in wild type (JC1352) and nse3-1 (JC3392). The enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) normalized to the negative control region as described in Fig 1B. (TIFF)

The nse3-1 mutants do not synchronize properly, however components of the Smc5/6 complex still interact. (A) Flow cytometry was performed as described in Fig 1. (B) The fold enrichment levels are relative to the late-replicating control region on Chr V for n = 3 experiments with the mean ± SD at the silent mating type locus (HMR) and two regions in...

TPE measurements from the URA3 reporter at Telomere VII L. TPE was determined in strains with the URA3 reporter at the adh4 locus of Chromosome VIIL. Overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates and photographed after incubation at 25C and 34C in wild type (JC1991), sir4Δ (JC3818), nse3-1(JC3860), nse3-1 sir4Δ...

Transcription at sub-telomeric genes in smc6-9 mutants. Levels of transcription were compared at sub-telomeric genes CHA1, VAC17 and YR043C as described in Fig 4 in wild type (JC470), sir4Δ (JC3737), smc6-9 (JC3039), and sir4Δ smc6-9 (JC3925). Expression values are mRNA levels relative to ACT1 and normalization to wild type cells. Error bars repres...

The nse3-1 allele, but not the smc6-9 allele shortens the long telomeres in cells lacking RIF2. Telomere length is determined for the indicated strains by performing southern blot analysis using radiolabeled poly GT/CA probe as explained in Fig 1F and in the experimental procedures section for wild type (JC470), rif2Δ (JC2992), smc6-9 (JC3039), and...

Comparison of ChIP levels for Smc6 at telomeres in sir4Δ and nse3-1 mutants and wild type cells. (A) Chromatin immunoprecipitation (ChIP) on Smc6FLAG in wild type (JC1594) and nse3-1 (JC2630) at 25°C. (B) ChIP comparison of Smc6FLAG in wild type (JC1594), sir4Δ (JC3732), nse3-1 (JC2630). The enrichment at three native subtelomeres (Tel1L, Tel6R and...

Disruption of epigenetic gene control mechanisms in the brain causes significant cognitive impairment that is a debilitating hallmark of most neurodegenerative disorders including Alzheimer's disease (AD). Histone acetylation is one of the best characterized of these epigenetic mechanisms that is critical for regulating learning and memory associat...

Expression levels of dTip60 at the larval NMJ. Figure (A) represents confocal imaging analysis of control w1118 larval boutons on muscles 6/7 at abdominal segment A4 immunohistochemically stained with dTip60 antibody (blue). Figure (B) dTip60 (blue) showing the presence of presynaptic dTip60 and absence of postsynaptic dTip60 localization due to RN...

Relative number of boutons to futsch stained loops. Histogram represents the relative number of boutons to futsch stained loops for each of the genotypes indicated calculated from data presented in Figure 3. (TIF)

Total number of boutons in control fly lines. (A) Flies homozygous for each of the genotypes indicated were crossed to flies homozygous for the nervous system elavC155 pan-neuronal GAL4 driver, and staged third instar progeny larvae were collected. Confocal imaging analysis of larval boutons on muscles 6/7 at abdominal segment A4 immunohistochemica...

a Test Cross Fly Lines. Ten flies homozygous for the dTIP60E431Q P-element insertion or control w1118 were mated to seven flies homozygous for the actin GAL4 driver line Act5c-GAL4: P{Act5c-GAL4}/CyO,y+). For independently derived fly lines dTip60E431Q A through D, the P-element insertions are located on chromosome 3. b Control Cross Fly Lines. Ten...

a Test Cross Fly Lines. Five male control w1118 flies, or five male flies containing dTip60RNAi P-element insertions, were mated to ten female virgin flies homozygous for the pan-neuronal elav- GAL4 driver located on chromosome X. The P-element insertion is located on the X chromosome for Dmel\TIP60/RNAi line A, and the second chromosome for lines...

a Probe set. b Listed is the gene name or CG accession number if the gene is uncharacterized. (DOCX)