zcchc8 cdna constructs  (Sino Biological)

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) Schematic representations of the NEXT complex and the minimal PAXT connection. To inactivate these pathways, ZCCHC8 and ZFC3H1 loci were targeted using CRISPR-Cas9. (B) Western blotting (WB) analysis of wild-type (WT) and three independent Zcchc8 −/− and Zfc3h1 −/− clonal cell lines (1–3). Blots were probed with the indicated NEXT- and PAXT-related antibodies and Vinculin (VCL, loading control). Non-specific antibody signals are indicated with an asterisk (*). (C) DE analysis of TE RNAs from Zcchc8 −/− (left) or Zfc3h1 −/− (right) cells versus their WT control. The x axes show the average log 2 FC of RNA-seq data, including multiple mappers ≤ 100, from three KO clones versus three WT samples, and y axes show the log 10 false discovery rate (FDR) values. Vertical lines denote log 2 FC = 0.5 or −0.5, and horizontal lines denote −log 10 FDR = 1. Red dots denote significantly upregulated values (log 2 FC > 0.5, FDR < 0.05), and gray dots denote significantly downregulated values (log 2 FC < −0.5, FDR < 0.05). The number (N) of significantly upregulated TE RNAs are indicated for each KO condition in red font. (D) Bar plots of upregulated TE RNAs stratified by class (x axis). The y axis shows the percentage of significantly upregulated (log 2 FC > 0.5, FDR < 0.05) RNAs relative to their genomic representation in Zcchc8 −/− and Zfc3h1 −/− versus control samples from multi-mapped (≤100) RNA-seq data. Absolute values of upregulated TE RNAs are indicated for each class. (E) As in (D) but stratified into retrotransposon subfamily classes. (F) Genome browser views of four upregulated TE RNA examples (LINE, LTR, SINE) from either unique or multi-mapped (≤100) RNA-seq data as indicated. RNAseq tracks from two replicates of stranded WT, Zcchc8 −/− , and Zfc3h1 −/− samples are displayed with relevant strand direction (+/−) and genomic coordinates (mm10). TE annotations are extracted from the mouse Repeatmasker genomic dataset (mm10). Gene models are based on Gencode (M22). (G) WB analysis showing depletion of 3F-mAID-tagged proteins in OsTIR1-HA-expressing cells following −/+ treatment with IAA (12 h). Samples were derived from untagged, Zcchc8–3F-mAID, Rbm7–3F-mAID , and Zfc3h1–3F-mAID cells. Membranes were probed with antibodies against ZFC3H1, FLAG, ZCCHC8, and Actin (ACTB, loading control). (H) qRT-PCR analysis of indicated NEXT ( proRPL27a ), PAXT ( SNHG10 ) or NEXT/PAXT ( proRNH1 ) targets or TE RNAs ( L1Tf , MuERV-L , B2 SINE ) from total RNA isolated from cells described in (G). Results were normalized to GAPDH mRNA levels and plotted relative to OsTIR1-IAA control samples. Columns represent the average values of technical triplicates (individual data as points) with error bars denoting the SD.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: CRISPR, Western Blot, RNA Sequencing Assay, Clone Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Isolation

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) Schematic representation of the HUSH complex. (B)WB analysis of FLAG IPs from chromatin lysates of WT and MTR4–3F cells. Chromatin input and IP samples were probed with antibodies against FLAG, ZFC3H1, MTR4, MPP8, and H3 (input loading control). (C) As in (B) but using ZCCHC8–3F cells. Membranes were probed with antibodies against FLAG, MPP8, MTR4, and H3 (input loading control). (D) WB analysis of MPP8 IPs from lysates of WT and TASOR-3F cells. IgG IPs were included as a negative control. Lysates from each cell line were split into two, with input samples loaded for each IP. Membranes were probed with antibodies against MPP8, FLAG, ZFC3H1, MTR4, ZCCHC8, and RPLP0 (input loading control). (E) WB analysis of MPP8 IPs from lysates of MPP8–3F-mAID or TASOR-3F-mAID cells either mock or IAA treated (8 h). Input and IP samples were probed with antibodies against MPP8, FLAG, ZCCHC8, MTR4, and RPLP0 (input loading control). (F) WB analysis of MPP8 IPs from lysates of TASOR-3F cells. Lysate extractions and IPs were carried out in increasing NaCl concentrations (0.1–1.0 M) as indicated. Membranes were probed with antibodies against MPP8, ZFC3H1, MTR4, ZCCHC8, FLAG, and Actin (ACTB, input loading control). (G) WB analysis of MPP8 IPs from WT lysates following mock or Benzonase treatment before final elution from beads. IgG IPs serve as a negative control. Lysates were split into two for either MPP8 or IgG IPs, with input samples loaded for each IP. Input and IP samples were probed with antibodies against MPP8, ZCCHC8, MTR4, TASOR, and TUBULIN (input loading control). (H) Metagene (upper) and heatmap (lower) profiles of unique mapped RNA-seq reads from WT, Zcchc8 −/− , and Zfc3h1 −/− datasets within a 10 kb window centered on MPP8 ChIP peaks. Heatmap rows are sorted by MPP8 peak signal intensities. Coverage of uniquely mapped reads are displayed for + and − strands. (I) Genome browser views of five MPP8 target loci. Displayed tracks include input and MPP8 ChIP-seq data from two replicate experiments as well as RNA-seq data from two replicates of WT, Zcchc8 −/− , and Zfc3h1 −/− samples. Strand directions (+/−) are noted along with genomic coordinates. TE hosting genes are indicated in parentheses.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: Negative Control, RNA Sequencing Assay, ChIP-sequencing

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) WB analysis of MPP8 IPs from lysates of WT, Z cchc8 −/− , Rbm7 −/− , Zfc3h1 −/− , and Zcchc8 −/− Zfc3h1 −/− cells. Input and IP samples were probed with antibodies against HUSH-, NEXT-, and PAXT-related proteins as indicated and Vinculin (VCL, input loading control). Non-specific bands are indicated with an asterisk (*). (B) WB analysis of MPP8 IPs from TET :: OsTIR1 - FLAG , MTR4 - 3F - mAID cells following doxycycline (DOX) and/or IAA treatment (4 h) as indicated. Input and IP samples were probed with antibodies against MPP8, MTR4, ZCCHC8 FLAG, and Actin (ACTB, input loading control). (C) Left: WB analysis of MPP8 IPs from WT or Zcchc8 −/− cells stably expressing MYC-tagged ZCCHC8 fragments labeled with amino acid numbers as in the right panel. Input and IP samples were probed with antibodies against MPP8, ZCCHC8, MYC, MTR4, and Vinculin (VCL, input loading control). Right: schematic representation of ZCCHC8 domains, generated fragments, and MPP8 IP data summary. Known protein binding regions are indicated on the top. Fragments shown to be HUSH binding (green) or not (red) are indicated, and a putative binding region is shown.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: Stable Transfection, Expressing, Labeling, Generated, Protein Binding, Binding Assay

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) Metagene (upper) and heatmap (lower) profiles of signals from input and MPP8 ChIP-seq samples from WT or Zcchc8 −/− datasets within a 2 kb window centered on MPP8 peaks. MPP8 ChIP samples are from two WT replicates and two Zcchc8 −/− clones. (B) Genome browser views of four MPP8-bound loci. Displayed tracks include input and ChIP-seq data from WT or Zcchc8 −/− cells as well as stranded RNA-seq data from the same cells. Only RNA-seq data from relevant strands are displayed as in . For intronic TEs, the relevant host gene is included in parentheses. (C) H3K9me3 ChIP-qPCR analysis at MPP8-bound loci ( Kcnq1ot1, Srrm2 ) and control regions not bound by MPP8 ( Pmp22 , Utp6 ) in WT or Zcchc8 −/− cells. Data are shown as the percentage of input with error bars indicating the SD of technical triplicates (individual data as points). Statistical significance was assessed using a two-tailed paired Student’s t test (*p < 0.05, **p < 0.01, ns, not significant). (D) qPCR analysis of IgG and FLAG ChIPs from MPP8-bound loci ( Cdc37l1 , Ncoa1 , Fgf14 ) and a control region not bound by MPP8 ( Utp6 ) in WT and Zcchc8 - 3F cells. Data are represented as percentage input values of three biological replicates and displayed as in (C). (E) WB analysis of lysates from WT and Mpp8 - mAID Zcchc8 - 3F OsTIR1 - HA cells either mock or IAA treated (12 h). Membranes were probed with antibodies against MPP8, ZCCHC8, FLAG, HA, and Actin (ACTB, loading control). (F) qPCR analysis of IgG and MPP8 ChIPs at MPP8-bound loci ( Kcnq1ot1 , Fgf14 , Cdc371l ) in from Mpp8 - mAID Zcchc8 - 3F OsTIR1 - HA samples described in (E). (G) qPCR analysis as in (F) but for IgG and FLAG ChIPs from the same samples.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: ChIP-sequencing, Clone Assay, RNA Sequencing Assay, Two Tailed Test

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) qRT-PCR analysis of L1 LINE transcripts from total RNA harvested from OsTIR1-HA , Zcchc8–3F-mAID , Mpp8 - 3F - mAID , or Zcchc8 −/− Mpp8 - 3 F- mAID cell lines either mock or IAA treated (72 h). Data representation as in . (B) WB analysis of lysates from three biological WT replicates and three Zcchc8 −/− clonal cell lines. Membranes were probed with antibodies against ZCCHC8, L1ORF1 and Actin (ACTB, loading control). (C) Quantification of L1ORF1 protein levels from the WB in (B). Data show the average value from three replicates, normalized to ACTB levels and plotted as the fold change relative to WT samples. Statistical significance was assessed as in . (D) WB analysis of Mpp8 - 3F - mAID , Zcchc8 −/− , and Zcchc8 −/− Mpp8 - 3F - mAID cell extracts following either mock or IAA treatment (72 h). Membranes were probed with antibodies against ZCCHC8, MPP8, L1ORF1, and RPLP0 (loading control). (E) Metagene profiles of 3′ end-seq signals from pA + and pA +,− 3′ end-seq libraries of WT or Zcchc8 −/− cells and displayed within a 10 kb window centered on MPP8 ChIP-seq peaks. Forward and reverse strands are plotted independently with replicates plotted separately as indicated in the legend. (F) Genome browser tracks of example upregulated L1 LINEs and LTR RNAs from RNA-seq data generated upon MPP8 or ZCCHC8 depletion. Data from Mpp 8- mAID samples, either mock or IAA treated (48 h), are from pA + selected, un-stranded libraries. Data from WT and Zcchc8 −/− cells are from rRNA-depleted, stranded libraries with the relevant strand data represented here. Annotations are displayed as in . (G) qRT-PCR analysis of L1Md_F transcripts from total RNA harvested from samples described in (A). Amplicons were designed to amplify either 5′, center, or 3′ regions of the L1Md_F2 LINE transcript as indicated in the schematics (H) As in (G) but for L1Md_T transcripts.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: Quantitative RT-PCR, ChIP-sequencing, RNA Sequencing Assay, Generated

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet: (A) The HUSH and NEXT complexes function to control expression of TE transcripts at either the transcriptional or post-transcriptional level, respectively. HUSH is recruited to TE loci decorated with H3K9me3 histone marks and is required for maintaining H3K9me3 levels and transcriptional (txn) suppression. NEXT is recruited to HUSH-bound loci through a physical connection that requires ZCCHC8 and MPP8. (B) In the absence of NEXT, HUSH can still bind to chromatin, regulate H3K9me3, and maintain low transcription levels. Without NEXT-mediated RNA decay, short pA − transcripts from TE loci are stabilized. (C) In the absence of HUSH, H3K9me3 levels are not maintained and NEXT is no longer recruited to HUSH-bound loci. TE loci lose transcriptional repression and show an increase in full-length pA + TE RNAs that, in the case of L1 LINEs, can be export competent and subsequently translated.

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: Expressing

Journal: Molecular cell

Article Title: Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression

doi: 10.1016/j.molcel.2022.03.004

Figure Lengend Snippet:

Article Snippet: ZCCHC8 cDNA constructs were cloned, using a full-length cDNA plasmid as a template (pUC19[mZCCHC8], Sino Biological), into a piggyBAC (pBAC) vector containing an N-terminal MYC tag and BSD selection marker using NEBuilder HiFi DNA assembly (NEB).

Techniques: Recombinant, SYBR Green Assay, Transfection, Clone Assay, Purification, Software

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Linkage analysis and whole-genome sequencing identify novel disease gene ZCCHC8 in familial pulmonary fibrosis with low telomerase RNA ( TR ). ( A ) Pedigree with pulmonary fibrosis proband (arrow) with affected relatives are indicated by the shaded symbols (key). The clinical history below each of the four shaded pedigree symbols refers to the age of onset of lung disease including idiopathic pulmonary fibrosis (IPF). (?) Asymptomatic individuals who had unknown affected status at the time of clinical assessment; (gray shading) unknown cause of death; (*) individuals with DNA who were included in the linkage analysis. ( B ) TR levels measured by quantitative real time PCR (qRT-PCR) in lymphoblastoid cell lines (LCLs). Arrow refers to proband (red) and pedigree identifiers refer to A . TR level from a DKC1 mutation carrier is a positive control. The data represent a mean of three experiments, each from independent RNA isolations. ( C ) Telogram shows age-adjusted lymphocyte telomere length by flow cytometry and fluorescence in situ hybridization (flowFISH) in the proband (arrow) and family (pedigree designations as in A ). The validated telogram is based on 192 controls. ( D ) Phenotype assignments used in linkage (key) and genotype below each individual refers to ZCCHC8 SNP. Italicized genotypes refer to obligate carriers. ( E ) Log of the odds (LOD) ratio across autosomal chromosomes calculated from SNP data from 14 individuals, with arrow on chromosome 12 pointing to maximum LOD. ( F ) p.P186L conservation across eight vertebrate ZCCHC8 species with darker shading denoting more conserved residues. CCHC refers to Zinc-knuckle domain; PSP refers to proline-rich domain.

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Positive Control, Flow Cytometry, Fluorescence, In Situ Hybridization

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 loss of function is sufficient to cause low TR levels. ( A ) Immunoblot of ZCCHC8 in lymphoblastoid cell lines (LCLs) from healthy controls (C1 and C2) and unaffected relatives and mutation carriers labeled with pedigree identifiers from A. Quantification from one blot and result replicated twice from independently harvested protein lysates. ( B ) Immunoblot of ZCCHC8, SKIV2L2, and RBM7 levels in proband's primary skin fibroblasts. ( C ) Immunoblot of transfected Myc-tagged (293FT cells) and endogenous ZCCHC8. ( D ) Mean ZCCHC8 mRNA levels ± SEM from LCLs in unaffected family members ( n = 4) and ZCCHC8 p.P186L mutation carriers ( n = 3). ( E ) Chromatogram showing that ZCCHC8 p.P186L mutation is expressed in LCL mRNA from proband (also verified in two other mutation carriers). ( F ) Immunoblot showing efficiency of shRNA knockdown of Luciferase (Luc), ZCCHC8, and NAF1 in HeLa cells. ( G ) Total TR levels measured by qRT-PCR (mean ± SEM from three independent knockdowns and RNA isolations). ( H ) Northern blot of TR after stable knockdown of ZCCHC8 and NAF1 (replicated twice with independent RNA isolations). (**) P < 0.01; (***) P < 0.001 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Mutagenesis, Labeling, Transfection, shRNA, Luciferase, Quantitative RT-PCR, Northern Blot

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended (>451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (<465 nt), and long genomically extended (>465 nt). Data are mean of three independent 3′RACE-seq analyses from three RNA isolations each from a different aliquot of a single clone. ( E ) qRT-PCR of extended TR forms beyond the 451 mature end (>20, >51, >784 nt). Data are mean of three independent RNA isolations similar to D . ( F ) TR Northern blot of edited ZCCHC8 +/+ and ZCCHC8 −/− cells. ( G ) Total TR levels by Northern bot (six blots from three RNA isolations). ( H ) Telomerase activity measured by telomere repeat amplification protocol (TRAP) assay in ZCCHC8 +/+ , ZCCHC8 −/− , and NAF1 S329/S329 HCT116 cell extracts. Activity was quantified on serially diluted extracts (1, 1/5, 1/25, and 1/125) against a PCR-amplified internal control (IC). RNase-treated wild-type extract and no template PCR reaction are included as negative controls. ( I ) Mean TRAP activity of 1/5× diluted extracts (three independent TRAP assays, each from a different lysate). ( J ) Summary of 3′RACE-seq of TR forms from control and proband's primary skin fibroblasts with speciation as in D . ( K ) qRT-PCR values of extended TR forms in primary skin fibroblasts as in E , mean of three technical replicates). ( L ) Amplified TR from input and Myc-ZCCHC8 immunprecipitated fractions (293FT cells) using primers falling within the mature TR sequence. ( M ) qRT-PCR of extended TR (>51 nt extended beyond the 3′ mature TR end) after transfection of tagged ZCCHC8, DIS3, EXOSC10/RRP6, and PARN into HCT116 ZCCHC8 −/− cells (three to four independent transfections/experiment). Data are expressed as mean ± SEM (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: CRISPR, Western Blot, Rapid Amplification of cDNA Ends, Sequencing, Quantitative RT-PCR, Northern Blot, Activity Assay, Amplification, TRAP Assay, Transfection

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Zcchc8- null mice have TR insufficiency. ( A – C ) Immunoblot for ZCCHC8, SKIV2L2, and RBM7, respectively, on lysates from mouse ear fibroblasts. ( D , E ) Northern blot for mouse TR and quantification. For E , mean reflects mice Zcchc8 +/+ ( n = 4, 2M/2F), Zcchc8 +/− ( n = 4, 2M/2F), Zcchc8 −/− ( n = 3M), mTR +/− ( n = 2, sex unknown) and mTR −/− ( n = 2, sex unknown). ( F ) TR 3′ extended levels (>20 bp) relative to Hprt as measured by qRT-PCR. Mouse numbers and M/F designations as in E . Data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Northern Blot, Quantitative RT-PCR

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 complete loss causes progressive and fatal neurodevelopmental phenotype. ( A , top row) Images showing head profile of Zcchc8 wild-type, heterozygous, and homozygous null mice (41–46 d-old). The labels show the genotype with a male ( left ) and a female ( right ) for each genotype. Zcchc8 −/− mice have abnormal head profiles with domed crania, as outlined by the dashed line. ( Middle row ) CT head mid-sagittal images show Zcchc8 −/− mice have dome-shaped crania. ( Bottom row). Volume-rendered (VR) CT images of mouse calvaria show widened cranial sutures in Zcchc8 −/− mice (seen in three of six imaged). None of Zcchc8 +/+ or Zcchc8 +/− mice (four mice imaged/genotype) had this feature. Each vertical image group is from the same mouse except the last column (two different females). ( B ) Representative H&E coronal sections from 8-wk-old heads (all male) show no differences in Zcchc8 +/− mice (11 examined) compared with Zcchc8 +/+ mice (10 examined). In contrast, Zcchc8 −/− mice had severe ventricular dilation (nine of 11 examined). ( C ) E12.5 brain sections show Zcchc8 −/− have microcephaly but intact brain structures with no ventriculomegaly in utero. ( D ) Image of E12.5 embryos from a single dam showing expected Mendelian ratios but Zcchc8 −/− embryos have small crania. ( E ) Cranial area of newborn (P0) pups measured on VR CT images and corrected to left femur length on the same images ( Zcchc8 +/+ n = 7, 3M/4F; Zcchc8 +/− n = 10, 3M/7F; Zcchc8 −/− n = 4, 3M/1F). ( F ) Mean cranial area ± SEM relative to age for all three genotypes showing that Zcchc8 −/− mice develop macrocephy after birth. Newborn mice include those listed in E and older mice ( Zcchc8 +/+ , n = 4, 3M/1F; Zcchc8 +/− , n = 4, 3M/1F; Zcchc8 −/− , n = 5, 3M/2F). Dashed lines denote 95% confidence intervals. (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: In Utero

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: The Zcchc8 −/− transcriptome shows up-regulation and misprocessing of low-abundance intronless RNAs other than TR . ( A ) Heat map with dendrogram of gene expression showing unsupervised analysis of 9788 high-quality genes from brain RNA-seq analysis. Colors denote mean-subtracted FPKM expression values on a log 2 scale ( Zcchc8 +/+ , n = 5; Zcchc8 +/− , n = 3; Zcchc8 −/− , n = 6 embryonic brains sequenced). Each column is labeled below by WT, HET, and KO followed by the embryo number (1, 2, 3, etc.), referring to respective Zcchc8 genotypes. The log 2 expression value was subtracted from the mean log 2 expression value of the entire cohort. The dendrogram showing relatedness of the samples is above , and relatedness of the gene transcripts is at the left . The differential change in RNA expression is shown as positive and negative change on color scale in the key above the top right corner. ( B , C ) Volcano plots depicting the log 2 -fold changes ( X -axis) versus −log 10 P -values calculated by two-tailed one-way ANOVA ( Y -axis) for the Zcchc8 +/− and Zcchc8 −/− versus Zcchc8 +/+ comparisons, respectively. Each dot represents a single transcript. ( D ) Histogram of number of genes at each expression value denoted on the x -axis by the mean log 2 FPKM values obtained from Zcchc8 wild-type embryos ( n = 5). RNAs that have more than two SD higher levels in the Zcchc8 +/+ versus Zcchc8 −/− comparison are shown in red ( n = 197) and fall on the low end of the histogram with TR and its mean FPKM in wild-type embryos shown. Down-regulated RNAs, defined as less than two SD ( n = 43), are shown in blue appear uniformly distributed on the distribution. ( E ) Histogram of the most up-regulated (>2SD) transcripts in the Zcchc8 −/− versus Zcchc8 +/+ by exon number shows the largest subset is intronless RNAs (42 of 188 with known gene structure, 22%). The pie chart divides the intronless RNAs by functional category. ( F ) Annotation of 28 up-regulated intronless RNAs ( TR , histones and cilia) shows a majority of the histones represented are replication-dependent histones (RDH) (23 of 24, 96%). The majority have an annotated transcript size in the range of TR between 400 and 560 (22 of 28). Columns referring to 5′ end and 3′ end refer to visualized additional reads beyond annotated gene boundaries with 5′ end reads referring to upstream reads that are not necessarily contiguous (manually identified in the Integrative Genome Viewer [IGV]). ( G , H ) Genome browser read coverage plots from IGV viewer showing extended 3′ ends as labeled above from two histone genes in each of Zcchc8 +/+ , Zcchc8 +/− , Zcchc8 −/− transcriptomes. ( I , J) Coverage plots for two coiled-coil domain containing cilia genes Ccdc89 and Ccdc182 , respectively, by genotype. For Ccdc89 , there is also an increase in discontinuous upstream of gene 5′end reads that resemble so-called promoter upstream transcripts (PROMPTs).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Expressing, RNA Sequencing Assay, Labeling, RNA Expression, Two Tailed Test, Functional Assay