C. elegans methionine/S-adenosylmethionine cycle activity is sensed and adjusted by a nuclear hormone receptor

Vitamin B12 is an essential micronutrient that functions in two metabolic pathways: the canonical propionate breakdown pathway and the methionine/S-adenosylmethionine (Met/SAM) cycle. In Caenorhabditis elegans, low vitamin B12, or genetic perturbation of the canonical propionate breakdown pathway results in propionate accumulation and the transcriptional activation of a propionate shunt pathway. This propionate-dependent mechanism requires nhr-10 and is referred to as “B12-mechanism-I”. Here, we report that vitamin B12 represses the expression of Met/SAM cycle genes by a propionate-independent mechanism we refer to as “B12-mechanism-II”. This mechanism is activated by perturbations in the Met/SAM cycle, genetically or due to low dietary vitamin B12. B12-mechanism-II requires nhr-114 to activate Met/SAM cycle gene expression, the vitamin B12 transporter, pmp-5, and adjust influx and efflux of the cycle by activating msra-1 and repressing cbs-1, respectively. Taken together, Met/SAM cycle activity is sensed and transcriptionally adjusted to be in a tight metabolic regime.


Introduction 28
Metabolism lies at the heart of most cellular and organismal processes. Anabolic 29 metabolism produces biomass during development, growth, cell turnover and wound 30 healing, while catabolic processes degrade nutrients to generate energy and metabolic 31 building blocks. Animals must be able to regulate their metabolism in response to nutrient 32 availability and to meet growth and energy demands. Metabolism can be regulated by Here, we report that vitamin B12 represses the expression of Met/SAM cycle 72 genes by a propionate-independent mechanism we refer to as "B12-mechanism-II". We 73 find that B12-mechanism-II is activated upon perturbation of the Met/SAM cycle, either 74 genetically or nutritionally, due to low dietary vitamin B12. This mechanism requires 75 another NHR, nhr-114, which responds to low levels of SAM. B12-mechanism-II not only 76 activates Met/SAM cycle gene expression, it also activates the expression of the vitamin 77 B12 transporter pmp-5 and the methionine sulfoxide reductase msra-1, and represses 78 the expression of the cystathionine beta synthase cbs-1. The regulation of the latter two 79 genes increases influx and reduces efflux of the Met/SAM cycle, respectively. These 80 findings indicate that low Met/SAM cycle activity is sensed and transcriptionally adjusted 81 to be maintained in a tightly controlled regime. Taken together, in C. elegans the genetic 82 or nutritional perturbation of the two vitamin B12-dependent pathways is sensed by two 83 transcriptional mechanisms via different NHRs. These mechanisms likely provide the 84 animal with metabolic adaptation to develop and thrive on different bacterial diets in the 85 wild. 86 87

Results 88
Low dietary vitamin B12 activates two transcriptional mechanisms 89 As in humans, vitamin B12 acts as a cofactor in two C. elegans pathways: the canonical 90 propionate breakdown pathway and the Met/SAM cycle, which is part of one-carbon 91 metabolism (Fig. 1A). These pathways are connected because homocysteine can be 92 converted into cystathionine by the cystathionine beta synthase CBS-1, which after 93 conversion into alpha-ketobutyrate is converted into propionyl-CoA. When   2019). Interestingly, we found that there is still a low level of GFP expression in Pacdh-114 1::GFP transgenic animals lacking nhr-10 (Fig. 1B). Since nhr-10 is absolutely required 115 to mediate the activation of acdh-1 by propionate, this means that there is another, 116 propionate-independent mechanism of activation. Importantly, the residual GFP 117 expression in Pacdh-1::GFP; Dnhr-10 was completely repressed by the supplementation 118 of vitamin B12 (Fig. 1B). This result was confirmed by inspecting our previously published 119 RNA-seq data: in Dnhr-10 animals there is residual endogenous acdh-1 expression which 120 is eliminated by the addition of vitamin B12 (Fig. 1C) (Bulcha et al. 2019). These results 121 demonstrate that there is another mechanism by which low vitamin B12 activates gene 122 expression that is independent of propionate accumulation, which occurs when flux 123 through the canonical propionate breakdown pathway is perturbed. We refer to the 124 activation of gene expression in response to canonical propionate breakdown 125 perturbation as "B12-mechanism-I" and the other, propionate-independent mechanism as 126 "B12-mechanism-II" (Fig. 1D)

Met/SAM cycle perturbations activate B12-mechanism-II 142
To determine the mechanisms by which "B12-mechanism-II" is activated, we used the 143 Pacdh-1::GFP vitamin B12 sensor in the Dnhr-10 mutant background, which cannot 144 respond to B12-mechanism-I. We first performed a forward genetic screen using ethyl 145 methanesulfonate (EMS) to find mutations that activate GFP expression in Pacdh-146 1::GFP;Dnhr-10 animals in the presence of vitamin B12 ( Fig. 2A). We screened ~8,000 147 genomes and identified 27 mutants, 16 of which were viable and produced GFP-148 expressing offspring. Seven of these mutants were backcrossed with the Pacdh-149 1::GFP;∆nhr-10 parent strain. Single nucleotide polymorphism mapping and whole 150 genome sequencing revealed mutations in metr-1, mtrr-1, sams-1, mthf-1 and pmp-5 ( Fig.  151 2B). The first four genes encode enzymes that function directly in the Met/SAM cycle (Fig.  152   1A). metr-1 is the single ortholog of human MS that encodes methionine synthase; mtrr-153 1 is the ortholog of MTRR that encodes MS reductase; sams-1 is orthologous to human 154 MAT1A and encodes a SAM synthase; and mthf-1 is the ortholog of human MTHFR that 155 encodes methylenetetrahydrofolate reductase. We also found mutations in pmp-5, an 156 ortholog of human ABCD4, which encodes a vitamin B12 transporter (Coelho et al. 2012). 157 Next, we performed a reverse genetic RNAi screen using a library of predicted 158 metabolic genes in Pacdh-1::GFP;Dnhr-10 animals fed E. coli HT115 bacteria (the 159 bacterial diet used for RNAi experiments) supplemented with vitamin B12 (Fig. 2C). In 160 these animals, GFP expression is off and we looked for those RNAi knockdowns that 161 activated GFP expression in the presence of vitamin B12. Out of more than 1,400 genes 162 tested, RNAi of only five genes resulted in activation of GFP expression: metr-1, mtrr-1, 163 sams-1, mthf-1 and mel-32 (Fig. 2D). Four of these genes were also found in the forward 164 genetic screen (Fig. 2B). The fifth gene, mel-32, also functions in one-carbon metabolism 165 (Fig. 1A). It is an ortholog of human SHMT1 and encodes serine 166 hydroxymethyltransferase that converts serine into glycine thereby producing 5,10-167 methylenetetrahydrofolate (5,10-meTHF), the precursor of 5-meTHF, which donates a 168 methyl group in the reaction catalyzed by METR-1 (Fig. 1A). These results show that 169 genetic perturbations in Met/SAM cycle genes activate B12-mechanism-II, even in the 170 presence of vitamin B12. This indicates that reduced activity of the Met/SAM cycle, either 171 due to genetic perturbations or as a result of low dietary vitamin B12 activates B12-172 mechanism-II ( Fig. 2E). Therefore, genetic perturbations in either pathway that uses 173 vitamin B12 as a cofactor activates the vitamin B12 sensor. 174 Giese et al., Figure 2 A B   Table S1). Importantly, 191 endogenous acdh-1 is upregulated in each of the Met/SAM cycle mutants, validating the 192 results obtained with the Pacdh-1::GFP vitamin B12 sensor (Supplemental Table S1).  Table S1). 197 The finding that Met/SAM cycle genes are transcriptionally activated in response 198 to genetic Met/SAM cycle perturbations implies that these genes may also be activated 199 by low vitamin B12. Indeed, inspection of our previously published RNA-seq data (Bulcha 200 et al. 2019) revealed that expression of Met/SAM cycle genes is repressed by vitamin 201 B12 (Fig. 3C). In contrast to propionate shunt genes, however, Met/SAM cycle genes are 202 not induced in response to propionate supplementation, nor are these genes affected in 203 nhr-10 or nhr-68 mutants, which are the mediators of the propionate response (B12-204 mechanism-I, Fig. 3D). Therefore, Met/SAM cycle gene expression is activated by B12-205 mechanism-II in response to either genetic or nutritional (low vitamin B12) perturbations 206 in the Met/SAM cycle (Fig. 3E). 207 Pacdh-1::GFP;∆nhr-10  We first tested the possibility that the accumulation of homocysteine in Met/SAM 312 cycle mutants may activate B12-mechanism-II. In C. elegans, RNAi of cbs-1 causes the 313 accumulation of homocysteine (Vozdek et al. 2012). Therefore, we reasoned that, if 314 homocysteine accumulation activates B12-mechanism-II, RNAi of cbs-1, should increase 315 GFP expression in the Pacdh-1::GFP vitamin B12 sensor. Remarkably, however, we 316 found the opposite: RNAi of cbs-1 repressed GFP expression in Pacdh-1::GFP;∆nhr-10 317 animals but not in the Met/SAM cycle mutants (Fig 5B; Supplemental Fig. S2A). This 318 indicates that a build-up of homocysteine is not the metabolic mechanism that activates 319 B12-mechanism-II. The repression of GFP expression by cbs-1 RNAi in Pacdh-320 1::GFP;∆nhr-10 animals could be explained by a decrease in the conversion of 321 homocysteine into cystathionine and an increase in the conversion into methionine 322 resulting in support of Met/SAM cycle activity (Fig. 1A). In sum, B12-mechanism-II is not 323 activated by a build-up of homocysteine. 324 Next we explored whether low methionine, low SAM or low phosphatidylcholine 325 activates B12-mechanism-II. We found that either methionine or choline supplementation 326 dramatically repressed GFP expression in Pacdh-1::GFP;Dnhr-10 animals (Fig. 5C). 327 However, neither metabolite greatly affected GFP levels in wild type reporter animals (Fig.  328   5C). Since these animals are fed vitamin B12-depleted E. coli OP50 bacteria and have 329 functional nhr-10 and nhr-68 TFs, GFP expression is likely high due to propionate 330 accumulation, i.e., B12-mechanism-I. Importantly, either methionine or choline 331 supplementation also repressed GFP expression induced by B12-mechanism-II due to 332 mutations in metr-1 or mthf-1 (Fig. 5D). However, while choline supplementation 333 repressed GFP expression in sams-1(ww51) mutants, methionine supplementation did 334 not (Fig. 5D). SAMS-1 converts methionine into SAM, and methionine levels are greatly 335 increased in sams-1 mutant animals, while being reduced in metr-1, mtrr-1 and mthf-1 336 mutants (Fig. 1A,5E). Since methionine levels are elevated in sams-1 mutants, and 337 because methionine supplementation cannot suppress GFP expression in these mutants, 338 these results indicate that low methionine is not the direct activator of B12-mechanism-II, 339 but rather that it is either low SAM, or low phosphatidylcholine, both of which require 340 methionine for their synthesis. In metr-1 and mthf-1 mutants methionine supplementation 341 supports the synthesis of SAM and phosphatidylcholine, and in these mutants, 342 methionine supplementation would therefore act indirectly. We did observe a mild 343 reduction in GFP levels upon methionine supplementation in sams-1(ww51) animals. This 344 is likely because it is not a complete loss-of-function allele, and/or functional redundancy 345 with three other SAMS genes. 346 To distinguish between the possibilities of low SAM or low phosphatidylcholine 347 activating B12-mechanism-II we next focused on pmt-2. PMT-2 is involved in the second 348 step of the conversion of phosphatidylethanolamine into phosphatidylcholine (Yilmaz and 349 Walhout 2016) (Fig. 1A). The expression of pmt-2 is repressed by vitamin B12, but not  Table S1). We 352 reasoned that pmt-2 RNAi might allow us to discriminate whether low SAM or low 353 phosphatidylcholine activates B12-mechanism-II. Specifically, we would expect pmt-2 354 RNAi to activate B12-mechanism-II if low phosphatidylcholine is the main cause, and 355 consequently that GFP expression would increase. Due to the severe growth delay 356 caused by RNAi of pmt-2 we diluted the RNAi bacteria with vector control bacteria. We 357 found that pmt-2 RNAi decreased GFP expression in Pacdh-1::GFP;Dnhr-10 animals 358 Met/SAM cycle activity results in low SAM levels, which activates B12-mechanism-II. 367 Next we asked whether nhr-114 is required for the transcriptional response to low 368 SAM. Since SAM is not stable and may not be easily absorbed by C. elegans, we used 369 methionine supplementation, which supports SAM synthesis, except in sams-1 mutant 370 animals. We performed RNA-seq in wild type and Dnhr-114 mutant animals with or 371 without methionine supplementation and found that Met/SAM cycle genes are repressed 372 by methionine supplementation in wild type, but not Dnhr-114 mutant animals (Fig. 5H,  373 Supplemental Table S3). Therefore, low SAM levels activate B12-mechanism-II in an nhr-374 114-dependent manner (Fig. 5I).

B12-mechanism-II actives influx and represses efflux of the Met/SAM cycle 398
Some of the most strongly regulated vitamin B12-repressed genes include msra-1 and 399 pmp-5 (Fig. 6A,B) (Bulcha et al. 2019). As mentioned above, pmp-5, which was also found 400 in our forward genetic screen, is an ortholog of human ABCD4, which encodes a vitamin 401 B12 transporter (Coelho et al. 2012). Thus, increased B12 transport may be used by the 402 animal as a mechanism to increase Met/SAM cycle activity. msra-1 encodes methionine 403 sulfoxide reductase that reduces methionine sulfoxide to methionine (Fig. 1A). This gene 404 provides an entry point into the Met/SAM cycle by increasing levels of methionine. This 405 observation prompted us to hypothesize that perturbation of Met/SAM cycle activity, either 406 by low dietary vitamin B12 or by genetic perturbations in the cycle, may activate the 407 expression of these genes. Indeed, both genes are induced in the Met/SAM cycle mutants 408 (Fig. 6A,B). Further, both genes are repressed by methionine supplementation, in an nhr-409 114 dependent manner (Fig. 6A,B). We also noticed that the expression level changes of 410 cbs-1 are opposite of those of msra-1 and pmp-5: cbs-1 is activated by vitamin B12, 411 repressed in Met/SAM cycle mutants, and activated by methionine in an nhr-114-412 dependent manner (Fig. 6C). As mentioned above, cbs-1 encodes cystathionine beta 413 synthase, which converts homocysteine into cystathionine (Fig. 1A). Reduced cbs-1 414 expression upon Met/SAM cycle perturbations would therefore likely prevent carbon 415 efflux. Finally, nhr-114 expression itself is repressed by both vitamin B12 and methionine 416 and activated by perturbations in Met/SAM cycle genes (Fig. 6D). This suggests that nhr- of B12-mechanism-I because it does not change when propionate is supplemented or 420 when nhr-10 is deleted. However, nhr-114 is mildly repressed in Dnhr-68 mutants. This 421 suggests that there may be some crosstalk between the two B12 mechanisms (Fig. 6D) 422 (Bulcha et al. 2019). Taken together, B12-mechanism-II is employed when Met/SAM 423 cycle activity is perturbed to increase Met/SAM cycle gene expression as well as 424 Met/SAM cycle activity and influx, and to decrease Met/SAM cycle efflux (Fig. 7). 425  Figure 7 Discussion 432 We have discovered a second mechanism by which vitamin B12 regulates gene 433 expression in C. elegans. This B12-mechanism-II is different from B12-mechanism-I, 434 which we previously reported to transcriptionally activate a propionate shunt in response  parental strain prior to sequencing. Genomic DNA was prepared by phenol-chloroform 537 extraction and ethanol precipitation. Fragmentation was carried out on a Covaris 538 sonicator E220 and 300-400 bp size fragments were collected using AMpure beads. 539 Libraries were prepared and barcoded using the Kapa hyper prep kit (KK8500). Samples 540 were sequenced at the core facility of the University of Massachusetts Medical school on 541 an Illumina HiSeq4000 using 50 bp paired-end reads. After filtering out low-quality reads, 542 300 million reads were recovered resulting in an 18X average coverage of the genome. 543 Reads were mapped to the C. elegans reference genome version WS220 and analyzed 544 using the CloudMap pipeline (Minevich et al. 2012) where mismatches were compared to 545 the parental strain as well as to the other sequenced mutants. Variants with unique 546 mismatches were validated by restriction fragment length polymorphism PCR (RFLP) and 547 sanger sequencing. 548 549

RNAi screen 550
RNAi screening was carried out as described (Conte et al. 2015). Briefly, RNAi clones 551 were cultured in 96 well deep-well dishes in LB containing 50 µg/ml ampicillin and grown 552 to log-phase at 37 o C. Clone cultures were concentrated to 20-fold in M9 buffer and 10 µL 553 was plated onto a well of a 96 well plate containing NGM agar with 2 mM Isopropyl β-d-  Animals were treated with NaOH-buffered bleach, L1 arrested and plated onto NGM 564 plates supplemented with 20 nM vitamin B12 and fed E. coli OP50. 400 late L4/early 565 young adult animals were picked into M9 buffer, washed three times and flash frozen in 566 liquid nitrogen. Total RNA was extracted using TRIzol (ThermoFisher), followed by DNase 567 I (NEB) treatment and purified using the Direct-zol RNA mini-prep kit (Zymo research). 568 RNA quality was verified by agarose gel electrophoresis and expression of known genes 569 were measured via RT-qPCR for quality control. Two biological replicates were 570 sequenced by BGI on the BGISEQ-500 next generation sequencer platform using 100 bp 571 paired-end reads. A minimum of approximately 40 million reads was obtained per sample. 572 Raw reads were processed on the DolphinNext RSEM v1.2.28 pipeline revision 7 573 The libraries were first demultiplexed by a homemade python script, and adapter 583 sequences were trimmed using trimmomatic-0.32 by recognizing polyA and barcode 584 sequences. Then, the alignment to the reference genome was performed by STAR with 585 the parameters "-runThreadN 4 --alignIntronMax 25000 --outFilterIntronMotifs 586 "c_elegans.PRJNA13758.WS271.canonical_geneset.gtf" were used as the annotation 590 table input for ESAT, but pseudogenes were discarded. The read counts for each gene 591 were used in differential expression analysis by DEseq2 package in R 3.6.3 (Love et al. 592 2014). A fold change cut off of greater than 1.5 and P adjusted value cut off of less than 593 0.01 was used. All the processing procedures were done in a homemade DolphinNext 594 pipeline. 595 The RNA-sequencing data files were deposited in the NCBI Gene Expression Approximately 100 synchronized L1 animals were plated across four wells of a 48 well 602 plate per condition. L4 animals were collected and washed three times in 0.03% sodium 603 azide and transferred to a 96 well plate. Excess liquid was removed, and plates were 604 rested for an hour to allow animals to settle and straighten. Pictures were taken using an 605 Evos Cell Imaging System microscope and image processing was done using a MATLAB 606 (MathWorks) script written in-house. 607 608 Gas chromatography-mass spectrometry 609 For Figure 5A, gravid adults were harvested from liquid S media cultures supplemented 610 with or without 64 nM vitamin B12 and fed concentrated E. coli OP50. For Figure 5D, 611 gravid adults were harvested from NGM agar plates treated with 64 nM vitamin B12 and 612 seeded with E. coli OP50. Animals were washed in 0.9% saline until the solution was 613 clear and then twice more (3-6 times total). Metabolites were extracted and analyzed as 614