a-robust-method-for-packing-high-resolution-c18-rp-nano-hplc-columns

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. ...

David Geffen School of Medicine at UCLA

DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM). Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 and DMS3, is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V.

A protein complex required for polymerase V transcripts and RNA- directed DNA methylation in Arabidopsis.

Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remodeling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3- and CHD1-stimulated gene activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator. In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.

Mediator coordinates PIC assembly with recruitment of CHD1.

Iron-sulfur (Fe-S) clusters are essential for many cellular processes, ranging from aerobic respiration, metabolite biosynthesis, ribosome assembly and DNA repair. Mutations in NFU1 and BOLA3 have been linked to genetic diseases with defects in mitochondrial Fe-S centers. Through genetic studies in yeast, we demonstrate that Nfu1 functions in a late step of [4Fe-4S] cluster biogenesis that is of heightened importance during oxidative metabolism. Proteomic studies revealed Nfu1 physical interacts with components of the ISA [4Fe-4S] assembly complex and client proteins that need [4Fe-4S] clusters to function. Additional studies focused on the mitochondrial BolA proteins, Bol1 and Bol3 (yeast homolog to human BOLA3), revealing that Bol1 functions earlier in Fe-S biogenesis with the monothiol glutaredoxin, Grx5, and Bol3 functions late with Nfu1. Given these observations, we propose that Nfu1, assisted by Bol3, functions to facilitate Fe-S transfer from the biosynthetic apparatus to the client proteins preventing oxidative damage to [4Fe-4S] clusters.

Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients.

Histone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.

A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development.

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in  that suppresses the transcriptional silencing of two  ( reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased  expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing.

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.

Mitochondria associate with lipid droplets (LDs) in fat-oxidizing tissues, but the functional role of these peridroplet mitochondria (PDM) is unknown. Microscopic observation of interscapular brown adipose tissue reveals that PDM have unique protein composition and cristae structure and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities; (2) PDM have reduced β-oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and β-oxidation; (3) PDM support LD expansion as Perilipin5-induced recruitment of mitochondria to LDs increases ATP synthase-dependent triacylglyceride synthesis; and (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis.

Mitochondria Bound to Lipid Droplets Have Unique Bioenergetics, Composition, and Dynamics that Support Lipid Droplet Expansion.

Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O) tension displaying a robust association in 21% O that is severely diminished in 1% O and contributes to O-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or carboxylate-modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange and reversed phase-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH reversed phase. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low-abundance bait proteins.

Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.

James A. Wohlschlegel

Department of Biological Chemistry

David Geffen School of Medicine at UCLA

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

James A. Wohlschlegel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Chemistry

David Geffen School of Medicine at UCLA

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns

Department of Biological Chemistry