X-linked oogenic transcripts are expressed late in the germline X-linked oogenic genes autosomal oogenic genes mex-3 Jones et al., 1996 rme-2 Almost no autosomal genes have an expression pattern restricted to the proximal germline; note these autosomal germline genes, which have expression in the distal part of the germline. Lee & Schedl, 2001

X-linked gene expression in the C. elegans germline

germline gene clustering on autosomes sperm and oogenic germline genes cluster in regional domains (p<10-15) Roy et al, Nature, 2002 “open” “closed”

operons in bacteria operons in worms

C. elegans operons 876 identified operons (>1000 extrapolated) 2270 genes (~15% of genome) 2.6 genes/operon average Unlike bacterial operons, operon genes in C. elegans do not show strong co-expression. (Blumenthal et al., 2002; Blumenthal and Gleason 2003) SL2 SL1

Operons show a chromosomal bias dots bars Blumenthal et al., 2002

Operons tend to encode proteins of certain functional classes Blumenthal and Gleason, 2003

Why are certain genes and not others in operons? C. elegans operons lack an organizing principle Relatively poor co-expression Low frequency of common functions for genes within an operon No functional class of proteins completely represented in operons Why are certain genes and not others in operons? There are two broad ideas for what selects for genes to be in operons. In the first, the genes in operons are more efficiently co-regulated than genes that do not share a single promoter and regulatory site. It is possible that the genes contained in operons need to be able to respond to global signals so they can be efficiently repressed or activated as a group. In the second broad class of explanation, the selection is simply for a compact genome; operons dramatically reduce both the DNA between genes and the amount of DNA expended on regulatory sites. Operons may mostly serve to transcribe genes that do not need to be regulated at all; they just need to be turned on in all tissues at all times. Another reasonable idea is that genes that are regulated at some level other than transcription have accumulated in operons because they can be expressed from the same promoter, but then regulated differentially at the level of mRNA stability or translation. These explanations are not mutually exclusive; different modes of selection may have resulted in the creation of different operons.

Similarities between germline and operon gene sets Show similar genomic organization: biased against the X Show similar frequency of nonviable phenotypes by RNAi: ~30% Encode similar types of gene products: oogenic germline set only

Operons contain similar functional classes as the oogenic germline gene set

Similarities between germline and operon gene sets Show similar genomic organization: biased against the X Show similar frequency of nonviable phenotypes by RNAi: ~30% Encode similar types of gene products: oogenic germline set only So, how many genes in operons are expressed in the germline?

# genes in operons on that chromosome

NextDB: Nematode EXpression paTtern DataBase Large-scale in situ hybridization distal+ proximal distal NextDB: Nematode EXpression paTtern DataBase Kohara lab Japan proximal distal

non-germline or no data 2283 genes in 876 operons Microarray: 32 spermatogenesis 1024 oogenic germline 1227 non-germline/ no data 1010 non-germline or no data 217 mildly germline 69 somatic 84 no data 1 132 germline 557 384 In situ: 96% of all genes in operons are expressed in the germline (1572/1642)

876 operons 510 completely classified operons (microarray or in situ data for ALL genes) 456 all-germline operons 54 mixed or somatic operons by in situ 89% of all operons are completely expressed in the germline (456/510)x100

Which came first? Expression: Germline gene expression actively promoted operon formation Function: Genes are in operons because of their encoded protein products, which happen to be commonly expressed in the germline. Test: look within a functional category for expression bias

Germline expression, not functional domains, determines operon formation 43 77 31 18 9 1 9 1 actin cytoskeleton 47% oogenic germline 90% germline operons protein phosphatases 26% oogenic germline 90% germline operons 14 that is, operons are correlated more tightly with gene expression in the germline than with the function of the encoded protein; indicating that germline expression was a more prominent force in the formation of operons 25 17 chaperones 75% oogenic germline 100% germline operons

Operons cluster in the genome 17/26 genes in operons 66kb from Chromosome II

Operons cluster in the genome chrom # operons # clustered % in clusters # clusters ave # in cluster CD p< I 192 120 63 41 2.9 1.30 0.0001 II 166 89 54 33 2.7 1.21 III 203 140 69 47 3.0 1.51 IV 151 74 49 28 2.6 1.24 0.0002 V 128 46 36 20 2.3 1.17 0.0010 X 6 17 3 2.0 1.07 0.1376 (25kb non-sliding window) Thanks to Scott Rifkin for writing statistics program for me

Operons cluster with monocistronic oogenic germline and sperm genes

Chromatin status in the germline “open” “closed” = operon = oogenic germline = sperm Sperm genes are expressed in the right place (the germline) and are are organized similarly to oogenic germline genes. Then why are they not in operons?

Oogenic germline genes have the shortest 5’ intergenic distances all operon (1st) all non-op oog gl non-op oog gl sperm non-op sperm mean 2051 1206 2215 1186 1468 1578 1602 SD 2964 1934 3080 2120 2501 2773 2802 median 972 539 1096 532 656 772 782 N 19484 870 17565 2901 1877 1278 1245

The REDUCE algorithm finds more putative regulatory elements in sperm genes than in oogenic germline genes Motif Gene # Significance AAGTCGCC 110 0.786 CACGTAAA 308 0.419 CGTGAACT 363 0.965 GATCTAGG 108 1.090 TTACGTGA 276 0.523 sperm oogenic germline ACTACGGT 242 0.465 CGCGCGAA 0.477*

Chromatin status in the germline “open” “closed” * * * * * * * * = operon = germline = sperm The requirement for sequence-specific transactivation (*) prevents operon formation, but the dependence on chromatin status and post-transcriptional regulation in the oogenic germline for gene regulation removes this requirement and allows operon formation when the trans-splicing machinery is present.