Data Set Group2: INIA Hypothalamus Affy MoGene 1.0 ST (Nov10)

Data Set: INIA Hypothalamus Affy MoGene 1.0 ST (Nov10) Male GN Accession: GN318 GEO Series: GSE36674 Title: Sex-specific modulation of gene expression networks in murine hypothalamus Organism: Mouse (Mus musculus, mm10) Group: BXD Tissue: Hypothalamus mRNA Dataset Status: Public Platforms: Affy Mouse Gene 1.0 ST (GPL6246) Normalization: RMA

Contact Information Robert Williams University of Tennessee Health Science Center 71 S Manassas St, Room 405 TSRB Memphis, TN 38163 USA Tel. 901 448-7050 rwilliams@uthsc.edu Website Download datasets and supplementary data files GN318_MeanDataAnnotated_rev081815.txt (N/A) GN318_StdError_rev081815.txt (N/A)

Specifics of this Data Set:

None

Summary:

These hypothalamic gene expression data were generated by Khyobeni Mozhui, Lu Lu, and Robert W. Williams and colleagues with funding support from NIAAA. The data set includes samples from 50 strains, including 46 BXDs, both parental strains, and reciprocal F1 hybrids. Expression data were generated using the Affymetrix Mouse Gene 1.0 ST exon-style microarray (multiple probes in all known exons) by Lorne Rose in the UTHSC Molecular Resources Center (MRC), Memphis TN. The table below provides a summary of cases, sex, and age. Hypothalamic tissue was dissected by K. Mozhui (description to follow) with special attention to time of day (every sample has time stamp). RNA was extracted by K. Mozhui. All other processing steps by the UTHSC MRC by L. Rose. Data were processed by Arthur Centeno. Data released initially Nov 25, 2010, updated March 7, 2011 by A. Centeno and K. Mozhui to add two additional arrays. Data appear to be error-free in terms of sex and strain assignments shown in the table below. Dissection protocol: Animals were sacrificed by quick cervical dislocation and brains were removed and stored in RNAlater (www.ambion.com) for 2 to 3 days Brain was placed with ventral side up and a partial cut was made with a blade at -2.5 from Bregma (just a little rostral from the pontine fibres when viewed from the ventral side) The brain was then place in a coronal matrix and a 2 mm section was made rostral to the first cut The 2mm Section was placed on a clean glass slide and hypothalamus was sliced out and placed in a tube on dry ice. To dissect out the BLA, the temporal lobes were detached by placing a scalple in the lateral ventricles and teasing it apart. The cortical amygdala was removed and the BLA was then sliced out and placed in a tube on dry ice. Tissues from two mice of the same strain and sex were pooled. The only exceptions to this are the BLA samples for strains BXD5, BXD13, BXD16, BXD19, BXD25, BXD38, BXD51, and BXD61 (tissue from only one animal). The hypothalamus contains nuclei and cell populations that are critical in reproduction and that differ significantly between the sexes in structure and function. To examine the molecular and genetic basis for these differences, we quantified gene expression in the hypothalamus of 39 pairs of adult male and female mice belonging to the BXD strains. This experimental design enabled us to define hypothalamic gene coexpression networks and provided robust estimates of absolute expression differences. As expected, sex has the strongest effect on the expression of genes on the X and Y chromosomes (e.g., Uty, Xist, Kdm6a).Transcripts associated with the endocrine system and neuropeptide signaling also differ significantly. Sex-differentiated transcripts often have well delimited expression within specific hypothalamic nuclei that have roles in reproduction. For instance, the estro-gen receptor (Esr1) and neurokinin B (Tac2) genes have intense expression in the medial preoptic and arcuate nuclei and comparatively high expression in females. Despite the strong effect of sex on single transcripts, the global pattern of covariance among transcripts is well preserved, and consequently, males and females have well matched coexpression modules. However, there are sex-specific hub genes in functionally equivalent modules. For example, only in males is the Y-linked gene, Uty, a highly connected transcript in a network that regulates chromatin modification and gene transcription. In females, the X chromo-some paralog, Kdm6a, takes the place of Uty in the same network. We also find significant effect of sex on genetic regulation and the same network in males and females can be associated with markedly different regulatory loci. With the exception of a few sex-specific modules, our analysis reveals a system in which sets of functionally related transcripts are organized into stable sex-independent networks that are controlled at a higher level by sex-specific modulators.

About the cases used to generate this set of data:

The BXD recombinant inbred strains are derived from crossing the C57BL/6J (B6) and DBA/2J (D2) parental strains and inbreeding for over 20 generations (Taylor et al., 1999; Peirce et al., 2004). All mice used in this study were born and housed at the University of Tennessee Health Science Center. Mice were kept at an average of 3–4 per cage in a temperature-controlled vivarium (22 deg C) and maintained at a 12 h light/dark cycle. All animal protocols were approved by the Animal Care and Use Committee. We studied a total of 50 BXD strains, but only acquired matched male–female data pairs for 39 strains (35 BXD strains, parental B6 and D2, and two reciprocal F1 hybrids, B6D2F1 and D2B6F1). The average age of mice was 78 days. We provide more detail on the experimen-tal design and precise age and time of sacrifice of all cases at stored in 4 deg C for 2–3 days. To dissect the hypothalamus, the brain was placed with the ventral side facing up in a coronal "brain cutting" matrix. Using the medial mammillary body as landmark, a vertical cut was made right along the posterior boundary of the hypothala-mus. A second vertical cut was made 2 mm from the first cut. This edge lies slightly caudal to the optic chiasm and cuts through the retrochiasmatic nucleus. The hypothalamus was then sliced out from this 2 mm thick section. Each of the 39 mouse strains is represented by male and female samples (total of 78 microarray samples). Each individual sample consisted of tissue pooled from two mice of the same strain and sex that are usually littermates. The total number of mice used was 78 females and 78 males. RNA was purified using the RNAeasy micro kit on the QIAcube system (Qiagen)2. RNA purity and concen-tration was checked using 260/280 nm absorbance ratio and RNA integrity was analyzed using the Agilent Bioanalyzer 2100 (Agilent Technologies). Index Array ID Tissue Strain Age Sex Date sacrifice Time sacrifice 1 R6854HYP Hyp C57BL/6J 77 F 8/18/10 8:45AM to 12:30 PM 2 R6862HYP Hyp C57BL/6J 77 M 8/18/10 8:45AM to 12:30 PM 3 R6852HYP Hyp D2B6F1 77 F 8/18/10 8:45AM to 12:30 PM 4 R6860HYP Hyp D2B6F1 77 M 8/18/10 8:45AM to 12:30 PM 5 R6864HYP Hyp DBA/2J 77 F 8/18/10 8:45AM to 12:30 PM 6 R6866HYP Hyp DBA/2J 68 M 8/18/10 8:45AM to 12:30 PM 7 R6858HYP Hyp B6D2F1 69 F 8/18/10 8:45AM to 12:30 PM 8 R6856HYP Hyp B6D2F1 69 M 8/18/10 8:45AM to 12:30 PM 9 R6800HYP Hyp BXD1 71 F 8/17/10 1:15 PM to 5 PM 10 R6796HYP Hyp BXD1 85 M 8/17/10 1:15 PM to 5 PM 11 R6788HYP Hyp BXD11 87 F 8/17/10 1:15 PM to 5 PM 12 R6786HYP Hyp BXD11 76 M 8/17/10 1:15 PM to 5 PM 13 R6806HYP Hyp BXD12 77 M 8/17/10 1:15 PM to 5 PM 14 R6812HYP Hyp BXD14 81 F 8/18/10 8:45AM to 12:30 PM 15 R6804HYP Hyp BXD24 85 F 8/17/10 1:15 PM to 5 PM 16 R6792HYP Hyp BXD27 75 F 8/17/10 1:15 PM to 5 PM 17 R6790HYP Hyp BXD27 73 M 8/17/10 1:15 PM to 5 PM 18 R6798HYP Hyp BXD29 71 F 8/17/10 1:15 PM to 5 PM 19 R6794HYP Hyp BXD29 71 M 8/17/10 1:15 PM to 5 PM 20 R6816HYP Hyp BXD31 74 F 8/18/10 8:45AM to 12:30 PM 21 R6802HYP Hyp BXD31 73 M 8/17/10 1:15 PM to 5 PM 22 R6916HYP Hyp BXD32 81 F 8/18/10 1 PM to 6:45 PM 23 R6846HYP Hyp BXD32 81 M 8/18/10 8:45AM to 12:30 PM 24 R6822HYP Hyp BXD34 77 F 8/18/10 8:45AM to 12:30 PM 25 R6808HYP Hyp BXD34 77 M 8/18/10 8:45AM to 12:30 PM 26 R6814HYP Hyp BXD39 79 M 8/18/10 8:45AM to 12:30 PM 27 R6848HYP Hyp BXD40 85 F 8/18/10 8:45AM to 12:30 PM 28 R6850HYP Hyp BXD40 85 M 8/18/10 8:45AM to 12:30 PM 29 R6810HYP Hyp BXD42 87 F 8/18/10 8:45AM to 12:30 PM 30 R6758HYP Hyp BXD43 81 M 8/17/10 9:30 AM to 12:30AM 31 R6746HYP Hyp BXD44 83 F 8/17/10 9:30 AM to 12:30AM 32 R6750HYP Hyp BXD44 83 M 8/17/10 9:30 AM to 12:30AM 33 R6764HYP Hyp BXD45 77 F 8/17/10 9:30 AM to 12:30AM 34 R6762HYP Hyp BXD45 77 M 8/17/10 9:30 AM to 12:30AM 35 R6880HYP Hyp BXD48 76 F 8/18/10 1 PM to 6:45 PM 36 R6882HYP Hyp BXD48 76 M 8/18/10 1 PM to 6:45 PM 37 R6748HYP Hyp BXD49 84 M 8/17/10 9:30 AM to 12:30AM 38 R6890HYP Hyp BXD50 77 F 8/18/10 1 PM to 6:45 PM 39 R6892HYP Hyp BXD50 77 M 8/18/10 1 PM to 6:45 PM 40 R6918HYP Hyp BXD56 84 F 8/18/10 1 PM to 6:45 PM 41 R6894HYP Hyp BXD56 77 M 8/18/10 1 PM to 6:45 PM 42 R6770HYP Hyp BXD60 70 F 8/17/10 9:30 AM to 12:30AM 43 R6772HYP Hyp BXD60 70 M 8/17/10 1:15 PM to 5 PM 44 R6836HYP Hyp BXD62 83 F 8/18/10 8:45AM to 12:30 PM 45 R6844HYP Hyp BXD62 83 M 8/18/10 8:45AM to 12:30 PM 46 R6888HYP Hyp BXD63 77 F 8/18/10 1 PM to 6:45 PM 47 R6878HYP Hyp BXD65 84 F 8/18/10 1 PM to 6:45 PM 48 R6874HYP Hyp BXD65 84 M 8/18/10 1 PM to 6:45 PM 49 R6930HYP Hyp BXD68 76 F 8/18/10 1 PM to 6:45 PM 50 R6932HYP Hyp BXD68 76 M 8/18/10 1 PM to 6:45 PM 51 R6776HYP Hyp BXD69 69 F 8/17/10 1:15 PM to 5 PM 52 R6774HYP Hyp BXD69 80 M 8/17/10 1:15 PM to 5 PM 53 R6926HYP Hyp BXD70 76 F 8/18/10 1 PM to 6:45 PM 54 R6922HYP Hyp BXD70 76 M 8/18/10 1 PM to 6:45 PM 55 R6870HYP Hyp BXD71 76 F 8/18/10 1 PM to 6:45 PM 56 R6872HYP Hyp BXD71 76 M 8/18/10 1 PM to 6:45 PM 57 R6778HYP Hyp BXD73 83 F 8/17/10 1:15 PM to 5 PM 58 R6780HYP Hyp BXD73 83 M 8/17/10 1:15 PM to 5 PM 59 R6838HYP Hyp BXD75 76 F 8/18/10 8:45AM to 12:30 PM 60 R6830HYP Hyp BXD75 76 M 8/18/10 8:45AM to 12:30 PM 61 R6934HYP Hyp BXD79 87 F 8/18/10 1 PM to 6:45 PM 62 R6782HYP Hyp BXD80 73 F 8/17/10 1:15 PM to 5 PM 63 R6784HYP Hyp BXD80 73 M 8/17/10 1:15 PM to 5 PM 64 R6914HYP Hyp BXD83 81 F 8/18/10 1 PM to 6:45 PM 65 R6912HYP Hyp BXD83 81 M 8/18/10 1 PM to 6:45 PM 66 R6834HYP Hyp BXD84 76 M 8/18/10 8:45AM to 12:30 PM 67 R6938HYP Hyp BXD85 74 F 8/18/10 1 PM to 6:45 PM 68 R6940HYP Hyp BXD85 74 M 8/18/10 1 PM to 6:45 PM 69 R6910HYP Hyp BXD87 83 F 8/18/10 1 PM to 6:45 PM 70 R6908HYP Hyp BXD87 83 M 8/18/10 1 PM to 6:45 PM 71 R6896HYP Hyp BXD89 82 F 8/18/10 1 PM to 6:45 PM 72 R6898HYP Hyp BXD89 82 M 8/18/10 1 PM to 6:45 PM 73 R6904HYP Hyp BXD90 82 F 8/18/10 1 PM to 6:45 PM 74 R6906HYP Hyp BXD90 82 M 8/18/10 1 PM to 6:45 PM 75 R6924HYP Hyp BXD92 86 F 8/18/10 1 PM to 6:45 PM 76 R6928HYP Hyp BXD92 89 M 8/18/10 1 PM to 6:45 PM 77 R6920HYP Hyp BXD95 76 F 8/18/10 1 PM to 6:45 PM 78 R6868HYP Hyp BXD95 76 M 8/18/10 8:45AM to 12:30 PM 79 R6900HYP Hyp BXD97 71 F 8/18/10 1 PM to 6:45 PM 80 R6902HYP Hyp BXD97 71 M 8/18/10 1 PM to 6:45 PM 81 R6876HYP Hyp BXD99 77 F 8/18/10 1 PM to 6:45 PM 82 R6884HYP Hyp BXD99 77 M 8/18/10 1 PM to 6:45 PM 83 R6840HYP Hyp BXD100 83 F 8/18/10 8:45AM to 12:30 PM 84 R6832HYP Hyp BXD100 83 M 8/18/10 8:45AM to 12:30 PM 85 R6944HYP Hyp BXD101 89 F 8/18/10 1 PM to 6:45 PM 86 R6942HYP Hyp BXD101 89 M 8/18/10 1 PM to 6:45 PM 87 R6756HYP Hyp BXD102 88 M 8/17/10 9:30 AM to 12:30AM 88 R6768HYP Hyp BXD103 78 F 8/17/10 9:30 AM to 12:30AM 89 R6766HYP Hyp BXD103 78 M 8/17/10 9:30 AM to 12:30AM

About the tissue used to generate this set of data:

About the array platform:

[MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST Array [transcript (gene) version] (GPL6246)

About data values and data processing:

Total RNA was processed using standard protocols and hybridized to the Affymetrix Mouse Gene 1.0 ST arrays4. This array contains 34,700 probe sets that target ∼29,000 well-defined transcripts (RefSeq mRNA isoforms). A single probe set is a collection of about 27 probes that target known exons within a single gene. The multiple probes design provides a more comprehensive cov-erage of transcripts from a single gene. Male and female samples were interleaved and processed together to avoid batch confounds. Details on the strain, age, and sex of each sample can be obtained from the information available for the INIA Hypothalamus Affy MoGene 1.0 ST (Nov10) data set on www.genenetwork.org. Probe set level intensity values were extracted from the CEL files using the Affymetrix GeneChip Operating Software. Data nor-malization was performed using the R package “Affy” available from www.Bioconductor.org. The Robust Multichip Averaging protocol was used to process the expression values. The array data was then log transformed and rescaled using a z-scoring procedure to set the mean of each sample at eight expression units with a SD of 2 units. The entire data set can be down-loaded from www.genenetwork.org using the accession number GN281 (http://www.genenetwork.org/webqtl/main.py?FormID = sharinginfo&GN_AccessionId = 281) and also from the NIH Gene Expression Omnibus5 using the GEO accession number GSE36674. For this study we used a subset of cases that were repre-sented by both male and female samples – 78 sex-balanced arrays. Statistical power provided by this sample size (N = 39 strains) was estimated using the R function power.t.test6 with the SD set at 0.17, which is the average value. Only transcripts with above aver-age expression (minimum expression value above 8.5 on a log2 scale) were included for further analysis (17,192 probe sets). We used a two-tailed paired t -test to identify transcripts with signifi-cant expression difference between males and females. We applied the Benjamini and Hochberg false discovery rate (FDR) method and selected differentially expressed transcripts using a 10% FDR criterion.

Notes:

Experiment Type:

Hypothalamus was dissected from adult male and female mice and process for expression analysis. RNA isolation Total RNA was purified using the RNAeasy micro kit on the QIAcube system (www.qiagen.com). RNA purity and concentration was checked using 260/280 nm absorbance ratio and RNA integrity was analyzed using the Agilent Bioanalyzer 2100 (Agilent Technologies). This preliminary data set is associated with 430 eQTLs with LOD scores above 10. Peak LRS is 146 for Trait ID 10513604 (Hdhd3).

Contributor:

Mozhui K, Lu L, Armstrong WE, Williams RW (2012) Sex-specific modulation of gene expression networks in murine hypothalamus. Front Neurosci. 2012 May 11;6:63. doi: 10.3389/fnins.2012.00063. eCollection 2012. Abstract The hypothalamus contains nuclei and cell populations that are critical in reproduction and that differ significantly between the sexes in structure and function. To examine the molecular and genetic basis for these differences, we quantified gene expression in the hypothalamus of 39 pairs of adult male and female mice belonging to the BXD strains. This experimental design enabled us to define hypothalamic gene coexpression networks and provided robust estimates of absolute expression differences. As expected, sex has the strongest effect on the expression of genes on the X and Y chromosomes (e.g., Uty, Xist, Kdm6a). Transcripts associated with the endocrine system and neuropeptide signaling also differ significantly. Sex-differentiated transcripts often have well delimited expression within specific hypothalamic nuclei that have roles in reproduction. For instance, the estrogen receptor (Esr1) and neurokinin B (Tac2) genes have intense expression in the medial preoptic and arcuate nuclei and comparatively high expression in females. Despite the strong effect of sex on single transcripts, the global pattern of covariance among transcripts is well preserved, and consequently, males and females have well matched coexpression modules. However, there are sex-specific hub genes in functionally equivalent modules. For example, only in males is the Y-linked gene, Uty, a highly connected transcript in a network that regulates chromatin modification and gene transcription. In females, the X chromosome paralog, Kdm6a, takes the place of Uty in the same network. We also find significant effect of sex on genetic regulation and the same network in males and females can be associated with markedly different regulatory loci. With the exception of a few sex-specific modules, our analysis reveals a system in which sets of functionally related transcripts are organized into stable sex-independent networks that are controlled at a higher level by sex-specific modulators. eQTL; hypothalamus; sex-specificity; weighted gene coexpression networks PMID: 22593731  PMCID: PMC3350311  DOI: 10.3389/fnins.2012.00063

Citation:

Mozhui K, Lu L, Armstrong WE, Williams RW. Sex-specific modulation of gene expression networks in murine hypothalamus. Front Neurosci 2012;6:63. PMID:22593731

Data source acknowledgment:

This work was supported by Integrative Neuroscience Initiative on Alcoholism grants U01AA13499, U01AA017590, U01AA0016662. The authors are also grateful to Arthur Centeno and Lorne Rose, and the Molecular Resource Center at UTHSC.

Study Id:

102

• The UT Center for Integrative and Translational Genomics
• NIGMS Systems Genetics and Precision Medicine project (R01 GM123489, 2017-2021)
• NIDA NIDA Core Center of Excellence in Transcriptomics, Systems Genetics, and the Addictome (P30 DA044223, 2017-2022)
• NIA Translational Systems Genetics of Mitochondria, Metabolism, and Aging (R01AG043930, 2013-2018)
• NIAAA Integrative Neuroscience Initiative on Alcoholism (U01 AA016662, U01 AA013499, U24 AA013513, U01 AA014425, 2006-2017)
• NIDA, NIMH, and NIAAA (P20-DA 21131, 2001-2012)
• NCI MMHCC (U01CA105417), NCRR, BIRN, (U24 RR021760)