Difficulties to accurately map epigenomes in a few cells sorted or dissected from tissues have hampered our understanding of how chromatin modification regulates development and diseases. Despite recent progress, all reported chromatin-immunoprecipitation-based deep sequencing (ChIP-seq) methods have not achieved high quality mapping of rare cell populations. We report Recovery via Protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq) for as few as 500 cells with superior quality compared to all reported techniques to date. FARP-ChIP-seq accurately mapped histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and multi-potent progenitors (MPPs) sorted from one mouse. These high quality datasets not only implicate genes involved in HSC differentiation but also demonstrate a general lack of H3K4me3/H3K27me3 bivalency on hematopoietic genes in HSCs. Thus the method offers accurate mapping for fewest cells. Overall design: two H3K4me3 replications for mESC, two to three replications of H3K4me3 and H3K27me3 for LT-HSC, ST-HSC and MPP

Difficulties to accurately map epigenomes in a few cells sorted or dissected from tissues have hampered our understanding of how chromatin modification regulates development and diseases. Despite recent progress, all reported chromatin-immunoprecipitation-based deep sequencing (ChIP-seq) methods have not achieved high quality mapping of rare cell populations. We report Recovery via Protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq) for as few as 500 cells with superior quality compared to all reported techniques to date. FARP-ChIP-seq accurately mapped histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and multi-potent progenitors (MPPs) sorted from one mouse. These high quality datasets not only implicate genes involved in HSC differentiation but also demonstrate a general lack of H3K4me3/H3K27me3 bivalency on hematopoietic genes in HSCs. Thus the method offers accurate mapping for fewest cells. Overall design: two H3K4me3 replications for mESC, two to three replications of H3K4me3 and H3K27me3 for LT-HSC, ST-HSC and MPP

We discovered that two mitotic regulators, BuGZ and Bub3, involved in splicing regulation during interphase Overall design: 8 samples from primary Human foreskin fibroblast cells (HFFs) , 12 samples from TOV21G cells. Control siRNA. BuGZ siRNA or Bub3 siRNA were transfected for 48 h before sample collection. Cells treated with pladienolide B served as positive controls. For each RNAi experiment, we had two biological replicates.

We discovered that two mitotic regulators, BuGZ and Bub3, involved in splicing regulation during interphase Overall design: 8 samples from primary Human foreskin fibroblast cells (HFFs) , 12 samples from TOV21G cells. Control siRNA. BuGZ siRNA or Bub3 siRNA were transfected for 48 h before sample collection. Cells treated with pladienolide B served as positive controls. For each RNAi experiment, we had two biological replicates.

We report the transcriptionalAresponseAof the zebrafish digestive organsAto an acute high-fat feed usingARNASeqAanalysisAand highlight the changes in geneAexpressionAinvolved in the synthesis, storage, and dispersal of lipids.AThese key physiological responses to a high-fat meal allAstem fromAthe endoplasmic reticulum (ER), where lipids are formed and assignedAtoAtheir fates. Overall design: A feeding time course was undertaken with 6.5-dpf larval zebrafish. Triplicate samples were independently prepared from pairwise crosses fed either high-fat or low-fat food. 5% egg yolk emulsion (high-fat) feeds and 10% egg white (low-fat) feeds were prepared. At the appropriate time points, digestive organs (intestine, liver, pancreas) were dissected from 10 anesthetized larval zebrafish. Unfed controls were used to determine a transcriptional baseline.

We report the transcriptionalAresponseAof the zebrafish digestive organsAto an acute high-fat feed usingARNASeqAanalysisAand highlight the changes in geneAexpressionAinvolved in the synthesis, storage, and dispersal of lipids.AThese key physiological responses to a high-fat meal allAstem fromAthe endoplasmic reticulum (ER), where lipids are formed and assignedAtoAtheir fates. Overall design: A feeding time course was undertaken with 6.5-dpf larval zebrafish. Triplicate samples were independently prepared from pairwise crosses fed either high-fat or low-fat food. 5% egg yolk emulsion (high-fat) feeds and 10% egg white (low-fat) feeds were prepared. At the appropriate time points, digestive organs (intestine, liver, pancreas) were dissected from 10 anesthetized larval zebrafish. Unfed controls were used to determine a transcriptional baseline.

Background: Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described.

Background: Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described.

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis.

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis.