Abstract
Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.
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Abbreviations
- MSL complex:
-
Male-specific lethal complex
- CLAMP:
-
Chromatin-linked adaptor for MSL proteins
- roX :
-
RNA on the X
References
Bai X, Alekseyenko AA, Kuroda MI (2004) Sequence-specific targeting of MSL complex regulates transcription of the roX RNA genes. EMBO J 23:2853–2861
Cai W, Jin Y, Girton J, Johansen J, Johansen KM (2010) Preparation of Drosophila polytene chromosome squashes for antibody labeling. J Vis Exp
Cugusi S, Kallappagoudar S, Ling H, Lucchesi JC (2015) The drosophila helicase Maleless (MLE) is implicated in functions distinct from its role in dosage compensation. Mol Cell Proteomics 14:1478–1488
Deuring R et al (2000) The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol Cell 5:355–365
Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196:961–971
Kelley RL, Solovyeva I, Lyman LM, Richman R, Solovyev V, Kuroda MI (1995) Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell 81:867–877
Kelley RL, Meller VH, Gordadze PR, Roman G, Davis RL, Kuroda MI (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98:513–522
Larschan E, Soruco MML, Lee O-K, Peng S, Bishop E, Chery J, Goebel K, Feng J, Park PJ, Kuroda MI (2012) Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation. PLoS Genet 8:e1002830
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
Lucchesi JC, Kelly WG, Panning B (2005) Chromatin remodeling in dosage compensation. Annu Rev Genet 39:615–651
Meller VH (2003) Initiation of dosage compensation in Drosophila embryos depends on expression of the roX RNAs. Mech Dev 120:759–767
Meller VH, Rattner BP (2002) The roX genes encode redundant male-specific lethal transcripts required for targeting of the MSL complex. EMBO J 21:1084–1091
Meller VH, Wu KH, Roman G, Kuroda MI, Davis RL (1997) roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell 88:445–457
Pimpinelli S, Bonaccorsi S, Fanti L, Gatti M (2000) Preparation and analysis of drosophila mitotic chromosomes. In: Sullivan W, Ashburner M, Hawley RS (eds) Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, p. 728
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Soruco MML et al (2013) The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation. Genes Dev 27:1551–1556
Venken KJT, He Y, Hoskins RA, Bellen HJ (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751
Villa R, Schauer T, Smialowski P, Straub T, Becker PB (2016) PionX sites mark the X chromosome for dosage compensation. Nature 537:244–248
Acknowledgements
We thank Dr. Koen Venken for providing us with the PacMan vector. We would also like to thank John Urban for the valuable input on experimental analysis and critical review of the manuscript. Additional thanks are given to Dr. Mitzi Kuroda and Dr. Peter Becker for providing antibodies. J.A.U. was supported by the National Institutes of Health award F31GM108423. All experiments were supported by the National Institutes of Health R01GM098461-1, American Cancer Society Research Scholar 123682-RSG-13-040-01-DMC, and a Pew Biomedical Scholars program grant.
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Figure S1
CLAMP amino acid sequence alignment from clamp mutants compared with wild type amino acid sequence. An alignment of the clamp 1 and clamp 2 amino acid sequences to the wild type CLAMP amino acid sequence shows the location of the two amino acid deletion generated by the clamp 1 allele (dashes in red). Red amino acids indicate the frame shift in the clamp 2 allele that results in an early stop codon. Amino acids in blue specify the difference between two CLAMP isoforms, one excluding (CLAMP-PA) and one including (CLAMP-PB) five amino acids. The six known zinc fingers are annotated with lines (PDF 51 kb).
Figure S2
Homozygous clamp 1 and clamp 2 larvae are present in decreased numbers compared with heterozygous siblings. (A) Over a period of ten days, a total of 212 clamp 1 larvae were collected. For each larva, the sex and clamp 1 genotype was determined. Plotted is the number of male and female heterozygous and homozygous clamp 1 animals collected at each time point. (B) Over a period of fourteen days, a total of 190 clamp 2 larvae were collected. For each larva, the sex and clamp 2 genotype was determined. Plotted is the number of male and female heterozygous and homozygous clamp 2 animals collected at each time point (PDF 66 kb).
Figure S3
Transcript abundance, protein levels and localization for CLAMP are unchanged in the clamp 1 mutants. (A) There are no significant changes in clamp transcript abundance in male or female clamp 1 heterozygous or homozygous mutant animals as measured by qRT-PCR. Shown is the average log2 fold change of three biological replicates after internal normalization to three reference genes (gapdh, rpl32, and ras64b). Each sex is normalized to the respective sex of the w −; clamp 2; P{CLAMP} rescue animal. (Error bars are +/− 1 S.E.M., **p < 0.01, *p < 0.05). (B) Protein was extracted from the salivary glands of clamp 1 heterozygous and homozygous males (left) and females (right) and Western blotted for CLAMP and Actin. Full length CLAMP (61 kDa) is produced in clamp 1 males and females (“C”). Below each graph is the quantification of each western blot. Plotted is the relative amount of CLAMP protein after internal normalization to actin and relative to y − w −. (C) Polytene chromosome immunostaining demonstrates there is no difference in CLAMP (green) localization of heterozygous or homozygous clamp 1 male and female larvae compared to respective y − w − wild type controls (PDF 374 kb).
Figure S4
Homozygous clamp 2 males die earlier in development than females. (A) DNA from first, second, and third instar larvae was tested for the Y-chromosome gene kl-5 to determine the presence or absence of male clamp 2 mutant animals. For first and second instar larvae, ten larvae were pooled together. The clamp 2 genotype of each sample is indicated above (“+” = wild type clamp, “-” = clamp 2 allele). The sex of the animals is also indicated above for third instar animals where it was possible to anatomically determine the sex of the animals. PCR of genomic DNA determined that kl-5 is present in pooled homozygous clamp 2 first and second instar larvae. Therefore, most homozygous clamp 2 male larvae die between the second and third instar larval stages. (B) Quantification of the anti-CLAMP Western blot in Fig. 2B shows the relative amount of CLAMP protein after internal normalization to Actin and relative to y − w −. There is no CLAMP protein produced in homozygous clamp 2 female larvae. (C) Mitotic chromosomes from female third instar brain neuroblasts are present in all conditions evaluated including clamp 2 null animals. The indicated insets below each panel show a 4X magnification of the boxed chromosomes. The top panels are optical stacks of 4 × 0.3 um each. Scale bar represents 20 μm (PDF 1187 kb).
Table S1
Primer sequences of targets tested by qRT-PCR (PDF 37 kb).
Table S2
clamp 1 and clamp 2 counts of larvae and adults eclosed. The number of clamp 1 and clamp 2 heterozyous and homozygous male and female larvae counted are shown (“Larvae” row). Of those larvae counted, the number of adults that eclosed is indicated (“Adults eclosed” row). The number in parentheses is the percent eclosed (PDF 45 kb).
Table S3
Chi-squared analysis for heterozygous and homozygous clamp 1 larvae. The number of observed (O) larvae for homozygous and heterozygous clamp 1 male or female larvae are indicated. The expected (E) number of each genotype of larvae was calculated using the total number of larvae counted (212). From these numbers, a Chi-squared statistic was calculated. To test for significance, the Chi-squared threshold was determined for 3 degrees of freedom (PDF 204 kb).
Table S4
Chi-squared analysis for heterozygous and homozygous clamp 2 larvae. The number of observed (O) larvae for homozygous and heterozygous clamp 2 male or female larvae are indicated. The expected (E) number of each genotype of larvae was calculated using the total number of larvae counted (190). From these numbers, a Chi-squared statistic was calculated. To test for significance, the Chi-squared threshold was determined for 3 degrees of freedom (PDF 204 kb).
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Urban, J.A., Doherty, C.A., Jordan, W.T. et al. The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females. Chromosome Res 25, 101–113 (2017). https://doi.org/10.1007/s10577-016-9541-9
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DOI: https://doi.org/10.1007/s10577-016-9541-9