Caenorhabditis Elegans Period Homolog Lin-42 Regulates the Timing of Heterochronic Mirna Expression

Caenorhabditis elegans period homolog lin-42 regulates the timing of heterochronic miRNA expression

Katherine A. McCulloch and Ann E. Rougvie1

Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455

Edited by Gary Ruvkun, Massachusetts General Hospital, Boston, MA, and approved September 23, 2014 (received for review August 3, 2014)

MicroRNAs (miRNAs) are small RNAs that regulate gene expression for review). Briefly, most miRNA genes are transcribed as long posttranscriptionally via the 3′ UTR of target mRNAs and were first primary transcripts (pri-miRNA) that are cleaved in the nucleus identified in the Caenorhabditis elegans heterochronic pathway. by the Microprocessor complex. This step generates an ∼70-nt miRNAs have since been found in many organisms and have broad precursor (pre-miRNA) with a hairpin structure that is exported functions, including control of differentiation and pluripotency in from the nucleus and processed by the Dicer endonuclease into humans. lin-4 and let-7–family miRNAs regulate developmental tim- an ∼22-nt heteroduplex. The mature miRNA strand from the ing in C. elegans, and their proper temporal expression ensures cell duplex is retained in a miRNA-induced silencing complex and lineage patterns are correctly timed and sequentially executed. Al- guides it to mRNA targets for silencing. Although much is though much is known about miRNA biogenesis, less is understood known about miRNA biogenesis, how this process is temporally about how miRNA expression is timed and regulated. lin-42,the sculpted to control development is less well understood. One gene worm homolog of the circadian rhythm gene period of flies and with a prominent role in timing miRNA expression is lin-28.The RNA-binding protein LIN-28 negatively regulates let-7 by blocking mammals, is another core component of the heterochronic gene – pathway. lin-42 mutants have a precocious phenotype, in which processing, a function that is conserved in mammals (9 13). In later-stage programs are executed too early, but the placement of addition, transcription factors encoded by the heterochronic genes lin-42 in the timing pathway is unclear. Here, we demonstrate that daf-12 and hbl-1 have been implicated in modulating miRNA transcription (14–16). However, given the complex temporal ex- lin-42 negatively regulates heterochronic miRNA transcription. let-7 pression patterns of heterochronic miRNA genes, there are likely and the related miRNA miR-48 accumulate precociously in lin-42 to be other factors involved in this process. mutants. This defect reflects transcriptional misregulation because LIN-42, the C. elegans homolog of the fly and mammalian cir- enhanced expression of both primary miRNA transcripts (pri-miRNAs) cadian clock protein Period (17), is a candidate regulator of het- and a let-7 promoter::gfp fusion are observed. The pri-miRNA levels erochronic miRNAs. Loss of lin-42 function causes a precocious oscillate during larval development, in a pattern reminiscent of lin-42 heterochronic phenotype in which the adult hypodermal program expression. Importantly, we show that lin-42 is not required for this occurs one stage too early, as opposed to the reiteration of larval cycling; instead, peak amplitude is increased. Genetic analyses fur- programs observed in miRNA mutants. In addition, lin-42 acts ther confirm that lin-42 acts through let-7 family miRNAs. Taken antagonistically to daf-12 to time cell fate decisions, and one role together, these data show that a key function of lin-42 in de- of daf-12 is to augment expression of let-7 family miRNAs (14, 15), velopmental timing is to dampen pri-miRNAs levels, preventing their suggesting that lin-42 might oppose miRNA function. premature expression as mature miRNAs. Here, we demonstrate that lin-42 is indeed a negative regulator of heterochronic miRNA expression. let-7 and miR-48 C. elegans | lin-42 | miRNA | let-7 | heterochronic accumulate precociously in lin-42(loss-of-function) [lin-42(lf)]

etazoan development uses both positional and temporal Significance Minformation to pattern life stages from the single-cell embryo to the reproductive adult. In Caenorhabditis elegans, the Caenorhabditis elegans studies have contributed greatly to temporal component of postembryonic patterning is conveyed our understanding of the temporal regulation of development. through the heterochronic gene regulatory circuit (see ref. 1 for These studies led to the discovery of microRNAs (miRNAs) review). A core theme of the heterochronic circuit is that a series and revealed that a succession of stage-specifically expressed of microRNA (miRNA) switches promote transitions from one miRNAs act as switches to guide life-stage transitions in cell stage to the next by negatively regulating key factors that direct fates. These miRNAs are conserved to humans and cause dis- stage-specific programs, thereby allowing successive temporal ease states when misexpressed, highlighting the importance of fates to be executed. their temporal regulation. Much is known about the biogenesis Five conserved miRNAs, lin-4 and the let-7 family members, and function of miRNAs, but how these processes are temporally let-7, miR-48, miR-84, and miR-241, play prominent roles in the sculpted is not well understood. This study reports that LIN-42, circuit (2–6). lin-4 appears first, midway through the L1 larval the C. elegans homolog of the circadian rhythm protein Period, stage and guides the transitionfromtheL1toL2stage(7). regulates timing of miRNA expression by modulating transcrip- miR-48, miR-84, and miR-241 amass in the L2 and control the tion. These findings further our understanding of the functions transition to the L3 stage, when let-7 levels rise dramatically and of developmental timekeepers and regulation of miRNAs. govern the switch to the L4. Mutations in these miRNAs cause the relevant transition to fail to advance. This process results in, Author contributions: K.A.M. and A.E.R. designed research; K.A.M. performed research; K.A.M. for example, repetition of the L1-stage pattern in lin-4 mutants contributed new reagents/analytic tools; K.A.M. and A.E.R. analyzed data; and K.A.M. or L2-stage patterns in mir-48/84/241 mutants, consistent with and A.E.R. wrote the paper. the sequential expression of these miRNAs. Deciphering this The authors declare no conflict of interest. temporal control mechanism then simplifies, to a large extent, to This article is a PNAS Direct Submission. understanding the timing of miRNA accumulation. 1To whom correspondence should be addressed. Email: [email protected]. miRNA biogenesis occurs via a multistep process that provides This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. many opportunities for controlling miRNA abundance (see ref. 8 1073/pnas.1414856111/-/DCSupplemental.

15450–15455 | PNAS | October 28, 2014 | vol. 111 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1414856111 Downloaded by guest on September 30, 2021 animals, and genetic analysis places these miRNAs downstream let-7 A 40 mlt-10 mRNA B 1200 of lin-42. The early appearance of these miRNAs likely reflects wild type wild type 35 transcriptional misregulation, because aberrant expression of lin-42(lf) 1000 lin-42(lf) 30 both pri-miRNAs and a let-7 promoter::gfp fusion are observed 800 25

in lin-42 mutants. Interestingly, the levels of all heterochronic 20 600

pri-miRNAs oscillate during larval development, in a pattern 15 400 Fold Change Fold Change reminiscent of lin-42 expression (17). Importantly, we show that 10 200 lin-42 is not required for pri-miRNA cycling. Instead, peak am- 5 0 0 plitude is increased in the absence of lin-42. Taken together, 12 16 20 24 28 32 36 40 44 48 52 56 18 22 26 30 34 38 42 46 50 54 Hours at 20°C Hours at 20°C these results indicate that a key role for lin-42 in developmental L1 L2 L3 L4 L2 L3 L4 timing is to dampen miRNA transcript oscillations, preventing L1 L2 L3 L2 L3 premature accumulation of mature miRNAs. CDL2-stage let-7 12 n.s. 300 wild type wild type 10 lin-42(lf) 250 lin-42(lf) Results ** 8 let-7 miRNA Accumulates Precociously in lin-42(ve11) Mutants. 200 miRNAs play prominent roles in the heterochronic gene path- 150 6 100 4 way, directing stage-to-stage transitions in temporal cell fates. Fold Change 50 2 Genetic data discussed above suggest that lin-42 could function to log2 Fold Change (let-7) 0 18hrs 28hrs 0 modulate accumulation of miRNAs that act in the timing pathway. L2 L3 To test this idea, let-7 levels were compared between synchronized 22hrs 32hrs populations of wild-type and lin-42(ve11) animals using Taqman Fig. 1. let-7 accumulates precociously in lin-42 mutants. (A) A representative miRNA quantitative RT-PCR (qRT-PCR) (Fig. 1 and Figs. S1 biological replicate of mlt-10 mRNA levels in wild type and lin-42(lf) mutants and S2). Because these populations are not directly comparable from 12 to 56 h after starved L1s were placed on food. All time points were based on absolute time from hatching, as a result of lengthening of normalized to wild type at 12 h. Here and in subsequent figures, approximate larval stages in lin-42 mutants (18), we used the cyclical expression stages based on mlt-10 expression are indicated with blue bars for wild-type pattern of mlt-10 as an internal control to align stages. Molting and red bars for lin-42(lf). (B) let-7 levels in wild type and lin-42(lf) mutants in defective-10,ormlt-10, encodes a nematode-specific protein that is the 18- to 56-h samples analyzed in A, normalizing to wild type at 18 h. The required for molting (19), and its message level peaks once per let-7 assay was specific and the data are representative of three biological larval stage in wild-type animals, ∼4 h before the onset of leth- replicates (Figs. S1 and S2). (C) The L2-stage data from B graphed using the argus at 20 °C (Fig. 1A). This temporal profile is similar to that mlt-10 profiles to align developmentally similar time points. (D) Graph of let-7 reported for a Pmlt-10::gfppest reporter (20) used to monitor levels at the time of mlt-10 peaks during the L2 and L3 stages averaged over three biological replicates. For example, the let-7 levels at 24 h in wild type and molting in individual lin-42 mutant animals (18). 28 h in lin-42(lf) were used from the biological replicate shown in A and B.For mlt-10 message levels also oscillate in lin-42(ve11) mutants statistical purposes, log2(fold-change) values are shown. **P = 0.002, n.s., not (Fig. 1A), but the period length increases, consistent with the significant, Welch’s t test. Error bars represent SD. observed developmental delay. Strikingly, lin-42(ve11) mutants only progress through three larval stages (with three corre- sponding mlt-10 peaks) in the time that wild-type animals com- type animals, again using mlt-10 expression to assess synchrony plete four and become adults. The mlt-10 peak time point in a and stage lengths. The wild-type expression patterns of the early- stage is considered to be developmentally similar between strains acting miRNAs closely match their profiles established by prior and is used as an alignment guidepost. Northern blot analyses (2, 5, 7, 22). lin-4 levels rise in the L1 lin-42(ve11) mutants were used for most of our studies because stage, whereas miR-48, miR-84, and miR-241 levels increase they are well characterized, have a strong heterochronic defect, later, rising dramatically in the L2 (Fig. 2A). and the mlt-10 profiles (Fig. 1A) indicate we can achieve a rea- Among the early-acting let-7-family miRNAs, only miR-48 lev- sonably high level of developmental synchrony, at least through els showed a reproducible and statistically significant precocious the early larval stages. lin-42(ve11), hereafter referred to as lin-42 increase in lin-42(lf) mutants (Fig. 2 and Fig. S3B), and this dif- (lf), contains a premature stop codon but is not a null allele (Fig. ference was most pronounced in the late L1 (Fig. 2B). We also S3A) (21). examined animals homozygous for a lin-42 null allele (lin-42(0) In wild-type animals, let-7 was not detectable until the animals (Materials and Methods) and obtained similar results (Fig. 2 A and approached the L1 molt, and remained low through the L2. B). lin-42(0) mutants have a strong molting defect, which results in During the L3 stage, let-7 levels rapidly increased (Fig. 1B) and remained high throughout subsequent development, in agree- L2 arrest of most animals, limiting the analysis to the L1 stage. lin-42 ment with previous northern analyses (2, 6, 13, 22). In contrast, miR-241 and miR-84 profiles were similar between in lin-42(lf) mutants, let-7 levels increased dramatically during mutants and wild type, aside from the developmental delay (Fig. BIOLOGY A C lin-4 the early L2 and continued to rise until the early to mid-L3, when 2 and ). miRNA levels increased dramatically during the DEVELOPMENTAL they became more equivalent to wild-type levels and were L1 stage in lin-42 mutants, as in wild-type animals (Fig. 2A), but somewhat variable (Fig. 1B). To more directly compare the let-7 were more variable than all other miRNAs examined. When expression pattern between strains, the data were aligned based averaged over multiple biological replicates, lin-4 levels were on mlt-10 expression and plotted to focus on the dynamics of the slightly higher in lin-42(lf) mutants versus wild type from the L1 L2 stage (Fig. 1 A and C). let-7 levels rose nearly 150-fold by the to the L2, but these differences were not statistically significant mid-L2 in lin-42(lf) mutants, whereas they remained just above (P > 0.05, two-way ANOVA) (Fig. S3B). Levels of three miRNAs background in wild-type animals. This result is reproducible and not in the heterochronic gene pathway did not change signifi- statistically significant across multiple biological replicates (Fig. cantly in L1-stage lin-42(lf) mutants (Fig. 2C). 1D and Fig. S3B). Therefore, one function of lin-42 is to temporally restrict let-7 levels during early larval development. pri-miRNA Transcript Levels Oscillate Through Larval Development. The miRNA biogenesis process provides multiple opportunities miR-48 Levels Are Increased in L1-Stage lin-42(lf) Mutants. Four for regulation. Because LIN-42 is nuclear (21) and related to additional miRNAs act earlier than let-7 in the timing pathway: period proteins of flies and mammals, which control gene ex- lin-4 and the let-7 miRNA family members miR-48, miR-241, pression by inhibiting transcriptional activators (23), one possi- and miR-84 (2, 4, 5). To test whether any of these miRNAs are bility is that the misregulation of let-7 and miR-48 observed in aberrantly expressed in lin-42 mutants, we focused on their dy- lin-42 mutants reflects transcriptional derepression. To test this namics in early larval stages when they are up-regulated in wild- idea, qRT-PCR assays were developed to track the primary

McCulloch and Rougvie PNAS | October 28, 2014 | vol. 111 | no. 43 | 15451 Downloaded by guest on September 30, 2021 whether lin-42 function is required for pri-miRNA cycling. Un- A miR-48 miR-241 1400 expectedly, pri-miRNA transcript levels oscillate in lin-42(lf) wild type 2000 1000 lin-42(lf) mutants, peaking once per larval stage, with a temporal profile lin-42(0) similar to that observed in wild type (Fig. 3A and Fig. S5). Be- 600 1000 cause hypomorphic lin-42 alleles leave one of three transcription units intact (Fig. S3A) (18, 21), it is essential to examine pri- Fold Change 200 0 6 1014182226303438 0 6 1014182226303438 miRNA levels in a null mutant background to rule out a role for L1 L2 L3 L1 L2 L3 LIN-42 function. Even in lin-42(0) mutants, heterochronic pri- L1 L2 L1 L2 L1 L1 miRNAs levels cycled once during the L1 stage (Fig. 3A and Fig. miR-84 lin-4 S5). Although we cannot exclude the possibility that the observed 80 600 profile was affected by the health of the strain, the majority of 60 these animals progress through the L1 and arrest development in 400 40 the late L2 stage. Thus, we conclude lin-42 activity is not required – – 200 to generate a cycle of pri let-7 or pri mir-48 expression. 20 Fold Change

0 0 6 1014182226303438 6 1014182226303438 L1 L2 L3 L1 L2 L3 L1 L2 L1 L2

L1 L1 Fold Change A pri-let-7 8000 BCL1-stage miR-48 L1 miRNA levels 1200 wild type 12 n.s. lin-42(lf) 6000 400 wild type 10 n.s. 800 * lin-42(lf) lin-42(0) 300 8 4000 lin-42(0) 200 6 400 n.s. Fold Change 2000 4 100 n.s. Fold Change 2 n.s. n.s. 0 6 10141822263034380

0 log2 Fold Change 0 L1 L2 L3 6hrs 18hrs 20hrs -2 miR-48miR-241miR-84 lin-4 miR-1miR-51miR-58 L1 L2 22hrs L1 pri-mir-48 Fig. 2. miR-48 levels are elevated in lin-42 mutants. (A) Fold-change in 300 levels of miR-48, miR-241, miR-84, and lin-4 in wild type, lin-42(lf),andlin-42(0) animals normalized to wild type at 6 h. The qRT-PCR assays were specific, and 200 the miRNA accumulation patterns were similar between biological replicates ≥ 100 (Figs. S1 and S2). Representative of 3 biological replicates, except for lin-42(0), Fold Change where n = 2. (B) Graph of miR-48 data for the L1 stage from A aligned for stage 0 6 1014182226303438 length. (C) Average miRNA levels during the L1 stage at the time point of L1 L2 L3 the mlt-10 expression peak (Fig. 1D and Fig. S5A). n = 3 biological replicates. L1 L2 *P < 0.05, n.s., not significant P > 0.05, Welch’s t test. L1 pri-let-7 B 3000 ) 2 1200 **

transcript levels of miRNA genes in the timing pathway (Fig. S1). 2000 800 The expression level of each pri-miRNA cycled during wild-type 1000 larval development (Fig. 3A and Fig. S4), generally peaking once 400 per stage. This finding is consistent with a previous study that 0 61014 18 22 Fold Change

Area (pixels 0 revealed oscillation of pri–let-7 levels (13). L1 wild typelin-42(lf) pri–let-7 and pri–lin-4 temporal profiles were similar to each L1 other (Fig. S4 A and B), with coordinate peak amplitudes at—or pri-mir-48 C 120 ) just before—each mlt-10 expression peak. The similar temporal 2 1200 * 80 patterns of lin-4 and let-7 transcription is surprising, given the 800 marked dissimilarity in the temporal accumulation of their miRNA 40 products (6, 7). mir-48 and mir-241 also exhibited comparable cy- 400 0 6 10141822 clical pri-miRNA patterns (Fig. S4 C and D). Given their relatively Fold Change 0 ∼ L1 Area (pixels wild typelin-42(lf) closeproximityinthegenome( 1.7 kb apart), coexpression of L1 these two miRNAs in a single primary transcript could be partly or n.s.

pri-lin-4 )

wholly responsible for the resemblance of their accumulation pat- D 2 1200 terns. However, there is some evidence for transcriptional con- 12 tributions from independent start sites (24). Finally, the expression 8 800 – – pattern of pri mir-84 differed from that of pri mir-48/241 (Fig. 4 400 S4E), even though there is temporal coordination in their mature 0 Area (pixels miRNA accumulation profiles (Fig. 2A). Although pri–mir-48/241 Fold Change 610141822 0 wild typelin-42(lf) – L1 levels peak during midlarval stages, pri mir-84 levels are highest L1 near the molt and peak out of phase with the other pri-miRNAs analyzed, suggesting that mir-84 is controlled by a different regu- Fig. 3. pri–let-7 and pri–mir-48 levels cycle and are increased in lin-42(lf) latory regime than that of its paralogs. mutants. All samples are normalized to wild type at 6 h. (A)pri–let-7 (Top) and pri–mir-48 (Bottom) levels in wild type, lin-42(lf), and lin-42(0) pop- Primary Transcript Levels Cycle in the Absence of lin-42. The ob- ulations. (B–D) pri-miRNA levels in L1-stage wild type and lin-42(lf) mutants served oscillations of heterochronic pri-miRNA levels are in- (Left). pri-miRNA expression during the L1 stage from ≥3 biological repli- triguing in light of the well-established cyclical lin-42 expression cates was calculated as the area under each curve in pixels-squared using pattern (17, 18, 21) and the role of period proteins in maintaining ImageJ software (Right) (40). (B)pri–let-7. (C)pri–mir-48. (D) pri-lin-4. Data the rhythmic expression of circadian-regulated genes in other graphed in B and C are technical replicates of that shown in A, except that organisms (25, 26). These relationships prompted us to ask 2× total RNA was used in the assay. **P < 0.01, *P < 0.05, Welch’s t test.

15452 | www.pnas.org/cgi/doi/10.1073/pnas.1414856111 McCulloch and Rougvie Downloaded by guest on September 30, 2021 pri–let-7 and pri–mir-48 Levels Are Increased in lin-42 Mutants. Al- wild type lin-42(lf) lin-42(lf) though lin-42 function is dispensable for generation of oscillatory AB C pri-miRNA expression patterns, peak amplitude was increased L2 for some pri-miRNAs in the mutants (Fig. 3 and Fig. S5). To 500 500 800 estimate the relative expression of each gene in wild-type versus DE F mutant animals, the areas under the L1-stage curves were compared. The L1 stage was chosen because during this stage, lin-42(lf) populations are most synchronous with wild type, and mis- L2m 500 500 800 expression of miRNAs is first apparent. A significant increase in the area under the pri–let-7 and pri–mir-48 curves was observed in lin-42(lf) compared with wild-type animals (Fig. 3 B and C), supporting a role for lin-42 in dampening let-7 and mir-48 transcription. In contrast, the area under the pri–lin-4 curve was GH I not significantly greater in lin-42(lf) than in wild type, similar to L3 – mature lin-4 miRNA levels (Figs. 2C and 3D). pri mir-241 ex- 800 800 350 pression was elevated in the L1 stage of some experiments, but this effect was not statistically significant when averaged over JKL several biological replicates (Fig. S5), similar to miR-241 levels. early L4 800 800 500 Plet-7::gfppest Expression Is Elevated and Precocious in the Seam of L3m lin-42(lf) Mutants. Although total pri–let-7 and pri–mir-48 levels are increased in lin-42(lf) mutants relative to wild type when synchronized populations are examined by qRT-PCR, a limitation of this approach is that transcript levels are averaged throughout Fig. 4. Plet-7::gfppest expression is elevated and detected early in lin-42 the animal. To simultaneously visualize the spatial and temporal pest dynamics of let-7 expression associated with loss of lin-42 function, mutants. Plet-7::gfp expression during midlarval development in wild-type we generated animals transgenic for an integrated Plet-7::gfp and lin-42(lf) animals with exposure times indicated (ms). (A and B) Expression reporter encoding a rapidly-degraded GFP variant (GFPPEST)(27) in the gut and pharynx of wild-type and lin-42(lf) animals, respectively. (C) GFP is precociously expressed in the seam of L2-stage lin-42(lf) mutants. (Inset) (SI Materials and Methods). The spatial pattern and onset of pest Magnification of two seam cells. GFP is not detected in the seam of wild type Plet-7::gfp expression in wild type was similar to previous at this stage. (D–F) Fluorescence (Top) and DIC (Bottom) images of L2-molt reports using standard gfp fusions (22, 28, 29), with expression stage animals. Plet-7::gfppest expression decreases during the L2 molt of wild observed in a variety of tissues (hypodermis, intestine, body wall type and lin-42(lf). Some GFP remains detectable in the pharynx and body muscle, pharynx, vulva, and nervous system) beginning in dif- wall muscle (BWM), particularly in the mutant. (G–I) Expression is detected in ferent larval stages. For example, gut and pharyngeal expression the seam of L3 stage wild-type animals and with greater intensity in lin-42(lf) began in the L1, whereas hypodermal seam cells, a site of rele- mutants. (J and K) Fluorescence (Top) and DIC (Bottom) images of L3-molt vance for the lin-42 heterochronic phenotype, were not observed stage animals. Plet-7::gfppest expression is not detected in the seam of L3m- until the L3 stage (Fig. 4 and Fig. S6). Notably, use of gfppest stage animals, although BWM and pharynx expression remains visible in the revealed cyclical expression in all tissues examined. GFP was mutant. (L) An early-L4 stage lin-42(lf) mutant lacks detectable GFP. (A–L) generally detected in multiple tissues late in each larval stage, Scale bar, 50 μm. but was weak or absent from molting animals (Fig. 4 D and J). Plet-7::gfppest expression cycled in lin-42(lf) mutants, with de- creased intensity in molting animals becoming undetectable by lin-42 mutants have a precocious phenotype in which adult cuticle the beginning of the next stage, similar to what was observed in is synthesized one stage early, during the L3 molt (17). In con- wild-type animals (Fig. 4 E, F, K, and L). However, two differ- trast, let-7 mutants and mir-48 mir-241 double-mutants have re- ences were apparent in lin-42 mutants. First, the intensity of GFP tarded phenotypes, inappropriately synthesizing larval cuticle fluorescence was elevated in lin-42(lf) mutants relative to wild during the final molt (2, 6). We analyzed lin-42(0); let-7(0) double- type in all tissues and at all stages where it was detected (Fig. 4 A, mutants and observed mutual suppression (Table 1), similar to B, G, and H), consistent with the increased levels of pri–let-7 experiments carried out with lin-42 hypomorphic alleles or lin-42 revealed by qRT-PCR. Second, Plet-7::gfppest expression was (RNAi) rather than a null (6, 21). At the L3 molt, the precocious detected precociously in hypodermal seam cells of all lin-42(lf) alae phenotype of lin-42(0) single-mutants was largely suppressed mutants analyzed (n = 22), appearing during the L2-stage, a time by loss of let-7, and furthermore, when alae were detected in the when pharyngeal and intestinal Plet-7::gfppest expression was double-mutant, they were weak and indistinct compared with readily evident in both mutant and wild-type animals (Fig. 4 B lin-42(0). At the L4 molt, the let-7(0) retarded phenotype was BIOLOGY

and C). On average, GFP was detected in 11.2 ± 3.4 seam cells strongly suppressed by the loss of lin-42; most animals had full- DEVELOPMENTAL per lateral side of late L2-stage lin-42(lf) animals (n = 8) but was length alae similar to wild-type animals. One interpretation of not detected at this stage in wild type (n = 15). Therefore, not this genetic result, supported by our molecular analysis, is that only does lin-42 play a global role in dampening let-7 transcrip- let-7 acts downstream but is not the only output of lin-42 activity. tion, but it also functions specifically in the L2 seam to prevent The three early-acting let-7 family members, mir-48, mir-241, let-7 expression. The early seam expression likely contributes to and mir-84, also promote temporal transitions in seam cells and the lin-42 precocious phenotype and helps explain the multiple have identical “seed” sequences to let-7, suggesting they may genetic interactions between lin-42 and heterochronic genes that share targets (2, 5) and could be additional outputs of lin-42 function in the L2 hypodermis, such as daf-12. activity. Although individual deletions of these miRNAs result in little or no heterochronic defect (2), we tested whether they let-7 Family Genes Act Downstream of lin-42 in the Timing Pathway. could suppress the lin-42(0) phenotype. lin-42(0); mir-241(0) and The increased levels of let-7 and miR-48 in lin-42 mutants sug- lin-42(0); mir-84(0) double-mutants are phenotypically similar to gest that these miRNAs are downstream of lin-42 in the heter- lin-42(0) mutants, indicating that misexpression of these miRNAs ochronic gene pathway, a hypothesis we tested with genetic makes little contribution to the lin-42 mutant phenotype (Table 1). epistasis. Mutations in heterochronic genes alter the timing of In contrast, deletion of mir-48 strongly suppressed the precocious hypodermal seam cell terminal differentiation, which occurs phenotype of lin-42(0) mutants. These results support our molec- during the final (L4) larval molt in wild type and results in gen- ular studies that revealed significant misregulation of mir-48 eration of characteristic adult cuticle containing ridges (“alae”). and let-7,butnotmir-241 or mir-84,inlin-42 mutants.

McCulloch and Rougvie PNAS | October 28, 2014 | vol. 111 | no. 43 | 15453 Downloaded by guest on September 30, 2021 Table 1. Genetic interaction of lin-42(0) and let-7-family genes Animals with L3-molt alae (%) Animals with L4-molt alae (%)

Genotype* Full Partial† None Quality‡ n Full Partial† None Quality‡ n

Wild-type (N2) 0 0 100 +++ 10 100 0 0 +++ 10 lin-42 66 32 2 +++ 87 ND ND ND ND let-7 unc-3 ND ND ND ND 11 26 63 + 38 lin-42; let-7 unc-3 01585++/+ 62 83 14 3 +++ 59 lin-42; unc-3 74 24 3 +++ 34 ND ND ND ND lin-42; mir-48 03565+++/+ 43 ND ND ND ND lin-42; mir-241 85 15 0 +++ 26 ND ND ND ND lin-42; mir-84 73 27 0 +++/++ 22 ND ND ND ND mir-48 mir-241 ND ND ND ND 2 88 19 ++ 42 lin-42; mir-48 mir-241 00100— 25 97 3 0 +++ 29 mir-48 mir-241; mir-84 ND ND ND ND 0 57 43 ++/+ 35 lin-42; mir-48 mir-241; mir-84 02 98+ 41 83 17 0 +++ 35 lin-42; mir-48 mir-241; let-7 unc-3 01387 + 30 19 22 59 + 37

ND, not determined. *The following null alleles were used: lin-42(ox461), let-7(mn112), mir-48 mir-241(nDf51), mir-48(n4097), mir- 84(n4037), mir-241(n4316). †Partial indicates that ≥1 of 16 seam cells scored per animal lacked alae in the overlying cuticle, resulting in gaps. ‡Alae varied in quality between strains but was of consistent quality within strains, from +++ (wild-type quality) to + (poor, indistinct alae quality).

The suppression of let-7(0) by lin-42(0) may reflect increased phenotype; hypodermal seam cells expressed the reporter early, mir-48 expression compensating for the absence of let-7. Indeed, beginning in the L2 rather than the L3 as in wild type. We did not overexpression of mir-48 from multicopy arrays can suppress detect significant increases in expression of the let-7-family let-7(0) (5). To test whether let-7 paralogs are required for members mir-84 and mir-241,orlin-4, although others found these suppression of let-7(0) by lin-42(0), we analyzed lin-42(0); nDf51; to be elevated in lin-42 mutants in late larval stages and adults let-7(0) mutants. nDf51 is a small deficiency that removes both (30, 31). Importantly, our epistasis analysis using null alleles mir-48 and mir-241 (2). At the L4 molt, the quadruple-mutant revealed that let-7 family miRNAs act downstream of lin-42, and exhibited a strong retarded phenotype similar to let-7(0) single- that misregulation of let-7 and mir-48 is the principle factor re- mutants (Table 1). Importantly, this genetic result demonstrates sponsible for the precocious hypodermal phenotype of lin-42 that lin-42 functions through these miRNAs to regulate devel- mutants. In the case of lin-4, we observed a trend for increased opmental timing; the absence of lin-42 does not alter seam de- accumulation in lin-42 mutants. However, lin-4 levels were vari- velopment when let-7 and mir-48 are also missing. Thus, genetic able relative to let-7 family miRNAs assayed in the same samples, analyses place let-7-family miRNAs, particularly let-7 and mir-48, and the differences were not statistically significant. pri–lin-4 downstream of lin-42 as key mediators of seam cell temporal fates. levels were also similar between the strains. It is possible that lin-4 levels are less sensitive to lin-42 mutation during early larval de- Discussion velopment because a Plin-4::gfppest reporter analysis showed the Five miRNAs play critical roles in regulating developmental greatest difference at the late L3–L3m stage (30). timing in C. elegans by accumulating sequentially and repressing Our work also adds to the increasing wealth of data showing earlier-stage gene-expression patterns to promote a succession of that the cyclical pri-miRNA expression profiles are often temporal cell fates. We demonstrate that lin-42, the C. elegans uncoupled from their mature miRNA patterns. This finding is homolog of the circadian rhythm gene period, negatively regu- particularly true of let-7: its primary transcript is strongly lates miRNA biogenesis, ensuring the appropriate expression of expressed during the mid-L1 stage, two stages before the accu- these temporal regulators. In lin-42 mutants, the primary tran- mulation of its miRNA. This uncoupling is in part because of scripts of let-7 and mir-48 are elevated, leading to precocious and action of the LIN-28 RNA-binding protein blocking processing enhanced accumulation of their miRNA products. The intensity (12, 13, 32). Therefore, lin-42 and lin-28 work in concert to of a Plet-7::gfppest reporter is increased in lin-42 mutants during regulate let-7, acting at the transcription and processing steps, larval stages, further supporting a model wherein LIN-42, similar respectively, and increased pri–let-7 levels in lin-42 mutants to period proteins, acts as a transcriptional repressor. could overwhelm the ability of LIN-28 to inhibit processing. The Our work complements and extends two recent reports (30, 31) L2 is likely to be particularly sensitive to the amount of pri–let-7 that also identify lin-42 as a negative regulator of miRNA tran- present because LIN-28 levels dramatically decline during this scription. The three studies reach this conclusion using diverse stage (33). Freed from inhibition, the excess pri-miRNA could approaches, analyzing a variety of mutant alleles and focusing on result in precocious accumulation of let-7. different larval stages. Our studies examine early stages (L1–L3), lin-42 also has a highly dynamic expression pattern with an interval when lin-42 function in the seam is supported by ge- mRNA and protein levels cycling once per larval stage and in netic interactions (21), whereas the other studies largely centered a broad variety of tissues (17, 18, 21). Given that LIN-42 is nu- on later stages, spanning the emergence of the hypodermal phe- clear, and that period proteins act as transcriptional repressors notype at the L3 molt in lin-42 mutants. Despite these differences, that program oscillatory expression patterns, LIN-42 was a prime each study concludes that dampening let-7 transcription is candidate to establish the pri-miRNA profiles. Testing whether a key output of LIN-42 function, indicating that this is not a stage- lin-42 is required for these cyclical patterns has been confounded restricted function, and the observed defects are not allele specific. by the fact that all described alleles leave one of three lin-42 Detailed analysis of early-stage dynamics uncovered precocious transcription units intact. We performed a definitive experiment accumulation of let-7 and miR-48 during the L1 and L2 stages. by examining pri-miRNA expression using a lin-42–null allele Temporal misregulation was also observed with a Plet-7::gfppest that deletes all transcription units. Surprisingly, the levels of the reporter in a tissue of relevance to the lin-42 heterochronic two pri-miRNAs examined, pri–let-7 and pri–mir-48, still cycled

15454 | www.pnas.org/cgi/doi/10.1073/pnas.1414856111 McCulloch and Rougvie Downloaded by guest on September 30, 2021 but their peak amplitudes were increased in the absence of lin-42. nematode growth medium (NGM) plates seeded with Escherichia coli OP50. Thus, lin-42 dampens, rather than programs, miRNA oscillations, let-7 suppression was not observed in this generation. Strain names and acting as a buffer to ensure that miRNAs are expressed at the details on genetics can be found in SI Materials and Methods and Table S1. appropriate levels and in the correct temporal window. The broad somatic expression lin-42, together with described Transgenic Strains and Fluorescence Microscopy. The Plet-7::gfppest reporter nonheterochronic functions, including control of molting and contains 3.2 kb of DNA extending 5′ of the pre-let-7 hairpin. Additional inhibition of dauer formation (18, 34), suggest its reach extends details about generation and analysis of transgenic animals can be found in beyond transcriptional regulation of heterochronic miRNAs. In- SI Materials and Methods. deed, genome-wide studies implicate lin-42 in modulation of both nonheterochronic miRNA genes as well as protein coding genes qRT-PCR. Starvation-synchronized L1s were grown on 10-cm OP50 seeded (30, 31), and it will be interesting to partition these targets among NGM plates at a density of ∼5,000 to ∼10,000 animals per plate. Biological lin-42 functions. Cyclical expression patterns linked to the molting replicates were performed with animals from independent hypochlorite cycle have emerged as a common theme in C. elegans larval de- treatments and grown on different days. Quantitative Taqman miRNA and velopment and include a large proportion of the transcriptome gene-expression assays were performed as directed (Life Technologies) using (35, 36), including the heterochronic miRNAs. An important goal Trizol to extract total RNA (Table S2). All quantitative PCR reactions were for the future is to decipher how these cyclical patterns are assayed in triplicate on an Eppendorf Realplex Thermocycler and Realplex generated and aligned with the molting cycles. Given the roles for 2.0 software. The sample used for normalization was run on every plate. U18 lin-42 in both molting and developmental timing, it may act to RNA was used as an internal control for miRNA experiments. ama-1 was used integrate these two developmental pathways. for normalization of pri-miRNAs and mlt-10 expression. Data were analyzed using the ΔΔCt method (39) and statistical analyses were performed using R. Materials and Methods Additional details can be found in SI Materials and Methods. Nematode Maintenance and Strains. C. elegans strains were cultured at 20 °C using standard methods (37). lin-42(ox461) is a null allele, generated by ACKNOWLEDGMENTS. We thank Dr. Tamar Resnick for thoughtful com- ments on the manuscript. Some strains were provided by the Caenorhab- mosDEL technology (38), in which the lin-42 coding region has been replaced + ditis Genetics Center, which is funded by NIH Office of Research Infra- with unc-119( ). lin-42(ox461); nDf51; let-7(mn112) unc-3(e151) animals structure Programs (P40 OD010440). This work was supported by Grant “ ” were maintained under conditions of hbl-1(low RNAi) (12), which R01GM50227 from the NIH (to A.E.R.); a grant from the Minnesota Med- weakly suppresses the lethality caused by let-7(0). To score, eggs were ical Foundation (to A.E.R.); and NIH Predoctoral Training Grant isolated from adults grown on low hbl-1(RNAi) plates and grown on 5T32HD007480 (to K.A.M.).

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McCulloch and Rougvie PNAS | October 28, 2014 | vol. 111 | no. 43 | 15455 Downloaded by guest on September 30, 2021