C. elegans maintenance and strains

Nematodes were grown with Escherichia coli OP50 bacteria on nematode growth medium (NGM) agar plates at 20 °C except where otherwise stated. All strains used in this study are listed in Supplementary Table 1.

Constructs and strains

Endogenously tagged rpc-1 at the C-terminus with STSGGSGGTGGS-mNeonGreen-GSAGSA-degron was obtained by CRISPR–Cas9 from the company SunyBiotech.

For the HIS-1–GFP fusion construct, the his-1 gene was amplified from N2 worm genomic DNA and fused by PCR to GFP, which contained introns. The ges-1 promoter and unc-54 3′ untranslated region were amplified from N2 genomic DNA. The final plasmid construct was generated by MultiSite Gateway cloning (Invitrogen). The strain expressing HIS-1/H4–GFP was made using the Mos1-mediated single-copy insertion technique76. The transgene was inserted into ttTi5605 on chromosome II.

The degron–GFP tagged rpoa-2 allele was constructed as described77 using Cas9 protein driven by eft-3 promoter in pDD162 and genomic RNA targeting a genomic sequence in the N-terminus of rpoa-2 in pRR13, a derivative of pRB1017, an empty vector for gRNA cloning. The degron–GFP-c1^sec^3xflag repair template was constructed for generating the knock-in into the N-terminus of the rpoa-2 gene. The 5′ and 3′ homology arms 751 bp upstream of the rpoa-2 start codon and 566 bp downstream of the start codon were used to replace the ccdB cassette in the degron–GFP-c1^sec^3xflag repair template. Each knock-in was isolated via hygromycin selection and the self-excising cassette (SEC) was then excised by heat shock to produce degron::GFP::rpoa-2 strain.

Information on the hetero- and euchromatic reporters used in Fig. 2 and Extended Data Fig. 2. In C. elegans, integrated transgene arrays acquire different chromatin marks and subnuclear locations, based on their size. Especially in embryos, large arrays that are composed of 300–500 plasmid copies are ‘heterochromatinized’ by the deposition of H3K9me3 and H3K27me3, peripherally located and silenced, while the same sequence, if present in less than 50 copies, is not78. The heterochromatin reporter used in Extended Data Fig. 2f–h, is a large array composed of about 300 copies. As cells differentiate, arrays carrying tissue-specific promoters shift inwards from the nuclear periphery exclusively in the tissue in which they are active, regardless of their copy number19. The euchromatic reporters used in Fig. 2d–f and Extended Data Fig. 2i–k are small transgene of about 30 copies19 driven by the pha-4 promoter, which is actively transcribed in intestine. Accordingly, these reporters are internally positioned in fed intestinal cells (ref. 19 and data in this study). The GW429 strain19 was created by ballistic transformation, generating a rare integration event of about 30 copies of the pha-4::mCherry plasmid. The 256 copies in the genotype refers to the copies of LacO repeats carried by the cointegrated plasmid to enable visualization of the allele by GFP–LacI.

Feeding, fasting and refeeding

Worms were maintained well fed on OP50 at 20 °C for at least two generations.

Fed L1 larvae (L1s) were obtained by washing plates of mixed-stage animals twice with M9 buffer to remove adults and larvae. The washes were performed with gentle swirling to avoid removing the bacteria. To obtain synchronized L1s, the embryos remaining on the plate were allowed to hatch for 2 h.

For L1s fasting, embryos were isolated by standard hypochlorite treatment and maintained in M9 buffer on a roller at 20 °C. L1s hatch approximately 12 h after hypochlorite treatment (ref. 79 and our own observation), and hence, this timepoint was considered 0 h of fasting. Consequently, 12 h of fasting corresponds to 24 h after hypochlorite treatment.

Refeeding was performed by placing 12 h fasted L1s on OP50-seeded NGM plates for the indicated time, after which they were collected for imaging.

To study the kinetics of chromatin reorganization during fasting, synchronized L1s were obtained as described above for fed L1s. They were then washed three times with M9 buffer for 10 min each and left in M9 buffer for the indicated time on a roller at 20 °C.

For fasting of adults, synchronized L1s by hypochlorite treatment were grown on OP50-seeded plates until day 1 of adulthood. Next, adults were collected with M9 buffer. A fraction of worms was immediately imaged for the fed state or dissected for intestine-specific RNA-seq, the rest were washed three times with M9 buffer for 10 min and fasted in M9 buffer for 12 h on a roller at 20 °C prior to imaging or intestine dissection.

For feeding in liquid, to obtain fed and synchronized L1s that were maintained in liquid for the same duration as the fasted animals, embryos obtained from hypochlorite treatment were kept in M9 buffer for 21 h. Next, L1s were pelleted, M9 buffer was removed and S-basal complete medium supplemented with 6 mg ml−1E. coli OP50 was added. L1s were subsequently fed in liquid culture for 3 h before imaging, reaching a total of 24 h in liquid.

For fasting on plates, isolated embryos obtained by hypochlorite treatment were placed onto M9 agarose plates without any bacteria for 24 h so that L1s were fasted for an average of 12 h as described above for fasting in liquid.

Auxin stock and plates

Auxin 3-indoleacetic acid (Sigma-Aldrich) was dissolved in ethanol to prepare a 57 mM stock solution and stored at 4 °C. Auxin was added to NGM plates to a final concentration of 1 mM. Control plates contained an equivalent amount of ethanol (1.75%). Plates were then seeded with OP50 bacteria.

Auxin treatment

For all degron-tagged strains, the tir1 allele ieSi57 was used except for experiments shown in Fig. 7a–e and Extended Data Fig. 7a–f, where the tir1 allele wrdSi23 was used.

Degradation of polymerase subunits

Degradation of polymerase subunits in strains expressing RPB-2-AID, RPC-1-AID and RPOA-2-AID was obtained as follows: fed L1s were obtained as described above and added onto auxin or ethanol plates containing Hoechst 33342-stained OP50 bacteria. L1s were maintained on auxin plates for the indicated amount of time, and on ethanol plates for 3 h, before imaging.

For RPOA-2 degradation in animals expressing HIS-72/H3.3–GFP, fed L1s were collected as described above and added onto auxins plates containing unstained OP50 bacteria.

To quantify the effect of RPB-2 depletion on the transcriptome (Extended Data Fig. 6e), animals were grown on OP50 at a density of approximately 40 animals per plate. Animals were maintained at 25 °C, which rendered the population sterile, and exposed to 375 µM of auxin (ɑ-naphthaleneacetic acid) starting at age 2 of adulthood. They were collected into lysis buffer for sequencing80 on day 6, 96 h post-auxin treatment.

For the refeeding experiments in absence of the polymerases core subunits, embryos were isolated using hypochlorite treatment and left in M9 buffer to allow L1s to hatch in absence of food. After a fasting period of 10 h (22 h post-bleaching), an aliquot of L1s was DNA live stained to verify the formation of the chromatin rings in intestine by microscopy, as follows: 10 h fasted L1s were incubated in M9 supplemented with 1 mM auxin and 40 µM Hoechst 33342 for 2 h in the dark, then L1s were imaged. To the remaining 10 h fasted L1s, auxin was added at a final concentration of 1 mM in M9 buffer. After 2 h on auxin (12 h of fasting), L1s were transferred to auxin plates seeded with OP50 bacteria stained with Hoechst 33342. Next, L1s were fed for 30 min and imaged.

For RPOA-2 degradation in adults, synchronized L1s by hypochlorite treatment were grown on OP50-seeded NGM plates for 72 h until day 1 of adulthood. Next, they were picked from the plates and transferred onto auxin plates seeded with OP50 and maintained for the indicated time.

Degradation of DAF-15

Embryos isolated by hypochlorite treatment were plated onto auxin, or ethanol plates as control, seeded with OP50 bacteria for 21 h. To stain the DNA, hatched L1s were moved onto auxin (or ethanol plates) seeded with Hoechst 33342-stained OP50 for 3 h. The absorption of auxin is not efficient in embryos due to their eggshell81. Thus, because L1s hatch approximately 12 h after hypochlorite treatment79, we considered our total 24 h of auxin treatment as embryos and L1s to correspond to 12 h of auxin exposure as L1s. Degradation of DAF-15-AID upon auxin treatment was previously performed and found to require 1 h on 1 mM auxin35.

Heat and cold stress

Fed L1s were placed onto standard NGM plates with OP50 and were subjected to either a 6 h heat shock at 34 °C or a 6 h cold shock at 6 °C in an air incubator.

Osmolarity

The final osmolarity of a solution was calculated by summing the osmolarity contributed by each solute present in the solution. To determine the osmolarity of each solute, the molarity of the solute was multiplied by the number of osmoles it produces when dissolved in water. M9 buffer was calculated to be 360 mOsm. To achieve different osmolarities, M9 buffer was diluted in MilliQ water to obtain buffers with osmolarities of 180, 150 and 120 mOsm. To create a solution of 540 mOsm, NaCl (90 mM) was added to M9.

Actinomycin D and α-amanitin treatments

A 25 mg ml−1 stock solution of actinomycin D (Bioaustralis) was prepared in dimethylsulfoxide. For the treatment plates, actinomycin D was diluted into NGM medium to achieve a final concentration of 100 μg ml−1. Control plates were prepared with an equivalent amount of dimethylsulfoxide (0.4%). The plates were seeded with OP50 bacteria and the bacteria were subsequently stained with Hoechst 33342 dye. Fed L1s were placed onto the plates and imaged after 6 h.

A 2 mg ml−1 stock solution of α-amanitin (Th. Geyer) was prepared in water. For the treatment plates, α-amanitin was diluted into NGM medium to achieve a final concentration of 25 μg ml−1. Control plates were prepared with an equivalent amount of water (1.25%). The plates were seeded with OP50 bacteria. Fed L1s were placed onto the plates and imaged after 6 h.

RNAi experiments

RNAi experiments were performed at 20 °C on NGM agar plates supplemented with 100 µg ml−1 carbenicillin and 400 µM isopropyl-β-d-thiogalactoside. Plates were seeded with double-stranded RNA-producing bacteria, grown overnight at 37 °C. As negative control, the RNAi clone containing the empty L4440 vector (Fire vector library) was used. In every experiment, let-607 RNAi was performed in parallel to confirm the efficiency of the RNAi plates, as a phenotype of very delayed growth/lethality is expected compared with worms grown on L4440 RNAi. RNAi clones were obtained from Ahringer library, Source BioScience Ltd. All RNAi clones used were sequenced to confirm target specificity.

For let-363 knockdown, synchronized L1s were seeded on RNAi plates and grown for 72 h. They were then transferred to freshly made RNAi-seeded plates. The following day, worms were washed off and discarded, leaving only laid embryos on the RNAi plates. After 2 h, hatched L1s were collected, washed three times, filtered with a 5 µm syringe strainer (PluriSelect) and seeded on fresh RNAi plates containing the respective RNAi bacteria. After 12 h, L1s of let-363 were collected for live imaging/RNA fluorescence in situ hybridization (FISH). To obtain stage-matched larvae, L1s seeded on the L4440 control were maintained on plates for 5 h before collection.

For mpk-1 knockdown, embryos isolated by standard hypochlorite treatment were placed and grown on RNAi plates until adulthood (P0). Next, the progeny of this RNAi-fed population was obtained by hypochlorite treatment of the adults and fasted for 12 h (24 h post-bleaching), before imaging.

For mes-6 and met-2, fasted animals were obtained as described for mpk-1 but fed L1s were also imaged in parallel by collecting the larvae that hatched on the RNAi plates.

For mes-6, RNAi efficiency was verified by incubating L1s on mes-6 RNAi plates for two generations, which produced 50–60% F1 adult sterility, as expected given the role of this gene in the germline82. For mpk-1, RNAi efficiency was verified by monitoring the amount of progeny of the RNAi-fed P0 and observing about 75% reduction, as expected given the role of this gene in the germline83.

Live staining of DNA

To prepare the staining solution, Hoechst 33342 (ChemCruz, 20 mM stock) was diluted 1:1,000 in M9 buffer, resulting in a final concentration of 20 µM. Next, the staining solution was added on OP50 bacteria, previously seeded and dried on NGM plates, making sure that all bacteria are covered. Next, the plates were left to dry in the dark.

To perform DNA live staining in fed larvae, L1s were obtained by washing plates of mixed stages and waiting 2 h for embryos to hatch. Hatched L1s were collected and added to these plates directly on the food and allowed to feed for 3 h on Hoechst 33342-stained bacteria.

Staining of the intestinal DNA in fasted worms was achieved by first feeding L1s with Hoechst 33342-stained bacteria for 3 h, as described above. Next, L1s were collected using M9, washed three times in M9 for 10 min to remove the bacteria and subsequently fasted in M9 buffer for 6–10 h.

Staining of DNA in fixed larvae

In Extended Data Fig. 2d, left, fasted L1s were fixed with 4% paraformaldehyde in PBS for 5 min and washed with PBS containing 30 mM glycine (PBS-G) for 10 min at room temperature. Next, worms were permeabilized with acetone at −20 °C for 1 min and subsequently washed with PBS-G. For staining, a solution was prepared by adding two drops of EasyProbe-Hoechst 33342 Live Cell Stain (GeneCopoeia) into 1 ml of M9 buffer. Next, the fixed worms were incubated in this staining solution for 15 min. After staining, a final 5 min wash with PBS-G was performed before preparing the samples for imaging on an agarose pad as described above but without sodium azide. In Extended Data Fig. 2d, right, fasted L1s were spotted on poly-l-lysine covered slides and allowed to settle for few minutes. Next, coverslips were applied and slides were snap frozen on dry ice for 25 min. To permeabilize worms, samples were then freeze-cracked by flicking the coverslips and immersed immediately in 100% ice-cold methanol for 10 s. Next, the slides were transferred to a fixing solution (0.08 M HEPES pH 6.9, 1.6 mM MgSO4, 0.8 mM EGTA and 3.7% formaldehyde, in PBS) for 10 min. After fixation, slides were washed three times with TBS-T (TBS with 0.1% Tween-20). To visualize DNA, samples were incubated with 10 µM Hoechst 33342 (ChemCruz) for 2 h, washed once with TBS-T, mounted with 80% glycerol in PBS and imaged.

rRNA FISH

To detect pre-rRNAs, we ordered six FISH probes targeting the internal transcribed spacer region 1 (its-1 in worms) labelled with fluorescent ATTO 488 at the 5′ end from Integrated DNA Technologies. Probe sequences are listed in Supplementary Table 3. To make the stock solution, each probe was dissolved in RNase-free H2O to a concentration of 1 μg μl−1. Next, 5 μl of each probe was pooled together and RNase-free H2O was added to reach a dilution of 1:12 for each probe (30 μl probes mix + 30 μl H2O). This stock solution was then diluted 1:500 in a hybridization buffer containing 2× SSC, 10% formamide, 5% dextran sulfate and 2 mM Ribonucleoside Vanadyl Complexes, to obtain the hybridization mix. Right before use, the hybridization mix was denatured for 5 min at 80 °C and incubated of at least 20 min at 37 °C.

Worms were collected and washed twice with PBS containing 0.01% Triton X-100 for 5 min each on a roller. They were then transferred to 1.5 ml tubes and pelleted at 1,500g for 3 min. The supernatant was aspirated, and 1 ml of fixation buffer (3.7% formaldehyde solution, 1× PBS and 0.01% Triton X-100) was added. The samples were incubated with fixation buffer for 45 min at room temperature. Afterwards, the samples were pelleted again, the fixation buffer was removed and the samples were washed twice with 1 ml of PBS containing 0.01% Triton X-100. Next, 1 ml of 70% ethanol was added and the samples were incubated overnight at 2–8 °C.

The next day, ethanol was removed after pelleting the samples and the fixed worms were rehydrated with PBS containing 0.01% Triton X-100 for 5 min at room temperature. The PBS was then removed, and 1 ml of permeabilization buffer (PBS with 0.5% Triton X-100) was added and incubated for 5 min at room temperature. After removing the permeabilization buffer, 1 ml of equilibration buffer (2× SSC, 10% formamide and 0.01% Triton X-100) was added and incubated for 15 min at room temperature. The equilibration buffer was then removed and the worms were incubated in the dark with hybridization buffer containing the final probe mix solution for at least 16 h at 42 °C while shaking at 1,200 r.p.m. in a thermal shaker.

The following day, after two brief washes with 2× SSC, the worms were incubated with wash buffer (1× SSC, 10% formamide and 0.01% Triton X-100) twice for 5 min each at 42 °C. This was followed by an incubation with Hoechst (1:2,000 in 1× PBS) for 30 min at room temperature. The worms were then washed again with wash buffer for 5 min at 42 °C and transferred to a poly-l-lysine-coated slide. To ensure proper adhesion to the surface, they were left to dry for a few minutes. Finally, the samples were mounted on microscope slides with Vectashield Plus Antifade Mounting Medium (Biozol), coverslips were applied and the edges were sealed with nail polish.

Microscopy

Microscopy was carried out using a live-cell imaging system (Confocal Spinning Disk Microscope) from Visitron Systems GmbH, equipped as follows: Nikon Eclipse Ti2 microscope with a Plan apo λ 100×/1.45 oil objective, Plan apo λ 60×/1.40 oil objective, Yokogawa CSU-W1 confocal scanner unit, VS-Homogenizer, Electron Multiplying CCD camera (Andor iXon Series) and VisiView software for acquisition.

Live microscopy was carried out on 2% agarose pads supplemented with 0.15% sodium azide (Interchim) to paralyse the worms, as previously described19. All Images were acquired with the Plan apo λ 100×/1.45 oil objective except for adults which were acquired with the Plan apo λ 60×/1.40 oil immersion objective. For each image, a range of stacks, from 50 to 120 stacks depending on the stage, were captured with a z-spacing of 200 nm.

Image analysesFluorescence intensities

Fluorescence intensities of GFP and mNeonGreen were measured using Fiji/ImageJ84,85.

For the polymerase subunits degradation experiments shown in Extended Data Figs. 5a–f and 7a, for each strain, images of the different treatments were acquired with the same settings. Intestinal nuclei were selected as region of interest based on Hoechst 33342 signal. Within each nucleus, quantitation of GFP or mNeonGreen mean signal intensity on focal stack images was done by selecting the brightest plane and subtracting the average background of the corresponding image.

For the analysis of RPOA-2-AID-GFP in whole larvae (Extended Data Fig. 7b), z-stack images were sum projected. Worms were selected as the region of interest using the freehand tool and the mean GFP signal intensity was measured. Background intensity was measured in animals expressing only TIR1 and subtracted from the GFP mean signal intensity.

For the RAGA-1GF–GFP experiment (Extended Data Fig. 6l), z-stack images were sum projected. Quantitation of the GFP mean signal intensity on the projected images was done by selecting the worms as the region of interest using the freehand tool and subtracting the average background intensity from the corresponding image.

Worm size measurements

For DAF-15 degradation, the experiment was conducted as described in ‘Auxin treatment’ section. The first images were acquired 24 h after plating the embryos on auxin and ethanol plates, which was considered day 1. For let-363 knockdown, the experiment details are described in ‘RNAi experiments’ section. Briefly, the first images were acquired immediately after transferring L1 larvae of the F1 generation onto fresh RNAi plates containing the RNAi bacteria. This was considered day 1. Images were captured with a Leica M165 FC fluorescent stereo microscope connected to a Leica K5 camera, using the LAS X software. Worm size was quantified using ImageJ. The freehand tool was used to draw around each worm and measure the area.

Chromatin profiling

Nuclei of mid-intestine, hypoderm and muscle were manually segmented in 2D or 3D using Cell-ACDC86 based on HIS-72–GFP or Hoechst 33342 signal (the latter works only for intestinal cells). To extract the intensity profiles shown in Figs. 1c,e,g,i, 2c, 3,c,f, 4b,h, 5b,g, 6c,d and 7b,e and Extended Data Figs. 1b,d,h,k, 3c,f,j,o, 4f, 6h,k, 7f and 8e, we implemented a custom routine written in Python. The analysis steps were as follows: (1) manual annotation of the centre of the inner dark area of the nuclei using Cell-ACDC. This is assumed to be the centre of the nucleolus. (2) Determination of the nuclei contours from the segmented objects (using the function from OpenCV package called ‘findContours‘). (3) Extraction of the intensity profiles from the centre determined in the previous step and all the points on the contour. (4) Normalization of each profile using the distance from the centre to the contour point (0% centre, 100% contour). (5) Binning the intensities into 5% width bins (for example, intensities at any distance in the ranges 0–5%, 5–10%, 10–15% and so on were considered being at 2.5%, 7.5%, 12.5% (bin centre) and so on distance from the centre). (6) Averaging of the binned normalized profiles along the distance to obtain the single-nuclei average profiles shown as single rows on the heat maps (Fig. 1b and Extended Data Figs. 1a,c,e,g,j l,m, 2c, 3b,e,h,i,n,p, 4a,c,e, 5g,h, 6c,g,j,n, 7d,e and 8d). (7) Normalization of each single-nucleus profile by its max intensity value. (8) Averaging and standard error calculation of the single-nuclei profiles within the same experimental conditions to obtain single-condition average profiles and its associated standard errors. Since 3D segmentation is quite time consuming, we set out to determine whether 2D segmentation would yield similar results. Therefore, we segmented the same nuclei in 3D and in 2D, where 3D segmentation allows for automatic determination of the centre z-slice (using the z coordinate of the 3D object’s centroid). We then compared the intensity profiles between 3D and 2D segmentation and found minimal differences. Thanks to this validation, we could proceed to segment all the other experiments in 2D.

To estimate the significance of the difference between the mean at each distance percentage 5% bin between chromatin profiles having a single peak, we performed permutation tests. To calculate a final P value, we adjusted the multiple P values for multiple tests using Bonferroni correction and combined the adjusted P values at three different regions of the plot (0–30%, 35–65% and 70–100%) using Pearson´s combined probability test.

To obtain the intensity profiles shown in Extended Data Fig. 2a, we applied the profile analysis described above but we included the nucleoli segmentation masks (see ‘Nucleolar volume analysis’ section). The centre of the inner dark area was therefore replaced with the centroid of the segmented nucleoli. Finally, the distances were not normalized as in (4), but they were plotted as absolute differences from the nucleolus edge (in μm).

Nucleolar volume analysis

Nuclei of mid-intestine, hypoderm and muscle were manually segmented in 2D using Cell-ACDC86 based on HIS-72–GFP or Hoechst 33342 signal and stacked into 3D ‘cylindrical’ objects. To detect and quantify the volume of nucleoli in 3D, we adapted the spot detection routine developed in ref. 87 and ref. 88. The analysis steps were as follows: (1) application of a 3D Gaussian filter with a small sigma (0.75 voxel) of the FIB-1::mCherry signal. (2) Instance segmentation of the spots’ signal using the best-suited automatic thresholding algorithm (either the threshold yen or Li algorithms from the Python library scikit-image89). (3) 3D connected component labelling of the thresholded mask obtained in (3) to separate and label the objects. (4) Manual inspection and removal of nucleoli segmented from other tissues. This step is required because nuclei were segmented in 2D and stacked into 3D, raising the possibility of nucleoli from other tissues to be detected. (5) The final volume of the nucleolus was calculated by fitting a 3D Gaussian function to the intensities of the pixels in the segmented mask. The 3D Gaussian function is

$$g\left(x,y,z\right)=g\left(x\right)g\left(\,y\right)g\left(z\right)+B,$$

where \(B\) is a fitting parameter and \(g\left(x\right)\), \(g\left(\,y\right)\) and \(g\left(z\right)\) are one-dimensional Gaussian functions given by

$$g\left(x\right)=A\exp \left(-\frac_}})}^}_}^}\right),$$

where \(_}}\), \(_\) and \(A\), are fitting parameters and they are the centre coordinate, the width (sigma) and the amplitude of the Gaussian peak. The \(_\), \(_\) and \(_\) parameters are then used to calculate the volume of the nucleolus with the following formula (ellipsoid’s volume):

Nucleolar/nuclear area

Mid-intestine nuclei were manually segmented in 2D in their central plane, and the area was calculated using Cell-ACDC86 based on HIS-72–GFP. Nucleoli were segmented in 3D as described in ‘Nucleolar volume analysis’ section. The segmentation mask was then projected to obtain a 2D representation and the area was calculated using ACDC.

rRNA FISH quantification

Nuclei of mid-intestine, hypoderm and muscle were manually segmented in 2D at their central plane using Cell-ACDC86 based on Hoechst 33342 signal. The segmentation mask was extended ten slices (of 200 nm each) above and below the central plane to cover the whole nucleus. To detect and quantify the FISH signal, we adapted the spot detection routine developed in ref. 87 and ref. 88. The analysis steps were as follows: (1) application of a 3D Gaussian filter with a small sigma (0.75 voxel) of the ATTO 488-labelled FISH probes. (2) Instance segmentation of the spots’ signal using the best-suited automatic thresholding algorithm (Li algorithms from the Python library scikit-image89). (3) 3D connected component labelling of the thresholded mask obtained in (3) to separate and label the objects. (4) The background-corrected sum of all the voxel values belonging to the segmented objects was calculated. Background correction was performed by subtracting the median of the pixels’ intensities outside of the segmented object and inside the nucleus at the central z-slice.

Chromatin rings analysis in adult and L1 larvae

The ratio between the outer and inner peak amplitudes of Extended Data Fig. 1n was calculated from each average single-nucleus chromatin profile (SNCP). To calculate the amplitudes, we fitted the following model to the SNCPs (sum of two Gaussian peaks)

$$B+}}_}\exp \left(-\frac_})}^}_}}^}\right)+}}_}\exp \left(-\frac_})}^}_}}^}\right),$$

where \(}}_}\), \(_}\), \(_}\), \(}}_}\), \(_}\), \(_}\) and \(B\) are fitting parameters. The subscripts \(}\) and \(}\) refer to inner and outer peaks, while \(}\), \(x\) and \(\sigma\) are the amplitude, centre location and standard deviation (a measure of the width) of the specific peak, respectively. The parameter \(B\) is the background level. The outer-to-inner ratios are therefore calculated as \(}}_}/}}_}\).

For the fitting procedure we used the Python function ‘scipy.optimize.curve_fit’ from the ‘scipy’ library90 with bounds on the fitting parameters. The amplitude and the background levels had a lower bound of 0 and an upper bound equal to the maximum of the SNCP; the centre location was bounded at ±5% (distance from the nucleolus centre of mass), and the standard deviation was bounded in the range (0%, 100%).

Resolution limit analysis

The procedure described in ‘Chromatin rings analysis in adult and L1 larvae’ section was also used to estimate the resolution limit of our imaging setup. The Gaussian function can in fact be used to approximate the Airy pattern91. Therefore, the average between σi and σo was used to compute the radius of the Airy disk as

$$d\cong 1.45_}+2\Delta _}},$$

where \(d\) is the minimum resolvable distance, \(_}\) is the average between \(_}\) and \(_}\), and \(\Delta _}}\) is the average distance between the centre of the peaks, \(_}\) and \(_}\), and the edges of the nucleolus and the nucleus, respectively. The nucleolus was segmented as explained in ‘Nucleolar volume analysis’ section.

Since muscle cells have the smallest space between nucleolus and nucleus, we wanted to compare single-nucleus profiles where non-resolvable radial profiles were not included. We estimated the minimum resolvable distance in muscle cells to be approximately 8.7 pixels (4.7 + 2 × 2 in the equation above); therefore, we calculated the single-nucleus profiles by discarding from the average those radial profiles whose nucleolar edge–nuclear periphery length was shorter than 9 pixels.

Positioning of heterochromatic and euchromatic reporters

Quantitation of heterochromatic (Extended Data Fig. 2h) and euchromatic reporter (Extended Data Fig. 2j) distributions on focal stacks of images was done with the ImageJ plugin PointPicker (http://bigwww.epfl.ch/thevenaz/pointpicker/) as previously described18. Briefly, for Extended Data Fig. 2h, the disc of the nucleus in which the spot is the brightest is divided into three zones of equal surface, each containing 33% of the area and the frequency of the spot in these three zones is quantified for fed and fasted nuclei. For Extended Data Fig. 2j, the relative nucleolus edge–nuclear periphery distance is divided into 10 bins, with 0–1 closest to the nucleolus edge and 9–10 closest to the nuclear periphery, and the frequency of the spot in each bin is scored for fed and fasted nuclei.

RNA extraction from L1 larvae and quantitative PCR with reverse transcription

To monitor the expression of pre-messenger RNAs in Extended Data Fig. 6d, the same number of RPB-2-degraded (3 h on 1 mM auxin) L1 larvae and TIR1-only controls was collected in Trizol (15596026, Thermo Fisher) and snap frozen in liquid nitrogen. After freeze cracking samples by five subsequent transfers from liquid nitrogen to 42 °C, samples were vigorously shaken for 2.5 min with 5× 30 s on/off cycles at room temperature. Next, total RNA was extracted following the manufacturer’s instructions of the Rneasy Mini kit (74104, Qiagen), including an on-column DNase digestion (79254, Qiagen).

Complementary DNA was obtained by using the Maxima H Minus cDNA Synthesis Master Mix (M1661, Thermo Scientific). For gene expression analysis, real-time PCR with the PowerUp SYBR Green Master Mix (A25742, Life Technologies) was used. Since the number of larvae was quantified and constant between the two samples, we performed a relative quantification, normalizing against unit mass by calculating the ΔCt against the control strain followed by the ratio (2ΔCt). All experiments were repeated at least three times. The primers are listed in Supplementary Table 3.

Intestine dissection

For fed adults, animals were transferred into a M9 bath in a 6 cm plate and washed for at least 10 min before intestine dissection, while fasted adults were directly used.

Adults were placed into a drop (~15 µl) of dissection buffer (M9 buffer with 0.1% Tween and 12 mM levamisole) on a glass slide. The intestine was dissected by decapitating the worm using Gr.20 syringes (Sterican), as previously described92. The extruding part of the intestine was cut off the worm carcass, then transferred into pre-cooled RL lysis buffer (Norgen Biotek Corp.) on ice. A total of 30 intestines was collected per condition.

RNA extraction for RNA-seq

RNA from dissected adult intestines was extracted using the Single Cell RNA Purification kit (Norgen Biotek Corp.) following the manufacturer’s instructions. RNA quality was assessed in an RNA Pico Chip on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the Qubit RNA HS Assay kit in a Qubit 3 Fluorometer (Life Technologies). cDNA synthesis, amplification and purification were performed using the SMART-Seq mRNA kit (Takara Bio) following the manufacturer’s instructions. In brief, 10 ng RNA was used as input for cDNA synthesis, 9× PCR cycles were performed for cDNA amplification and cDNA was purified using AMPure XP beads (Beckman Coulter) in a ratio of 0.6:1 to cDNA. cDNA quality was assessed on a High Sensitivity DNA Chip on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay kit in a Qubit 3 Fluorometer (Life Technologies). Dual-indexed libraries were prepared using the NexteraXT kit (Illumina) and sequencing was performed on a NextSeq2000 (Illumina) sequencer.

To quantify absolute transcript levels (Extended Data Fig. 6e), 30 nematodes were picked per replicate for a total of four technical replicates per sample and added to a lysis buffer, which was prepared together with a mix of External RNA Controls Consortium spike-ins93. cDNA libraries were prepared as previously described94.

RNA-seq analysis

For Fig. 8, reads were analysed as described previously95. In summary, adaptors were trimmed using Trimmomatic v0.39. Reads were aligned to the C. elegans genome (ce10) with Bowtie2 v2.4.5 and the R package QuasR v1.42.1,(www.bioconductor.org/packages/2.12/bioc/html/QuasR.html). The command ‘proj <-qAlign(‘samples.txt’,‘BSgenome. Celegans.UCSC.ce10’, splicedAlignment=TRUE)’ instructs hisat296 to align using default parameters, considering unique reads for genes and genome-wide distribution. Count tables of reads mapping within annotated exons in WormBase (WS220) were constructed using the qCount function of the QuasR package to quantify the number of reads in each window (qCount(proj, GRange_object, orientation = ‘same’)) and normalized by division by the total number of reads in each library and multiplied by the average library size. Transformation into log2 space was performed after the addition of a pseudocount of 8 to minimize large changes in abundance fold change (FC) caused by low count numbers. The EdgeR package v4.0.14 was applied to select genes with differential transcript abundances between indicated conditions (contrasts) based on false discovery rates (FDR) and log2 FC. The intestinal specificity of all samples (Extended Data Fig. 8b,g) was tested by measuring the expression enrichment within available datasets of different worm tissues96. Pearson coefficients describing the correlation of replicas are shown in Extended Data Fig. 8a,f. GO terms were extracted from wormbase.org and the GO term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed using the gprofiler2 package (v 0.2.2) as an interface to g: Profiler97.

For Extended Data Fig. 6e, differences in absolute transcript abundance between samples were calculated via differential expression analysis using DESeq2 (ref. 98), with DESeq2 size-factors calculated exclusively from reads mapping to ERCC spike-ins.

Statistics and reproducibility

No statistical method was used to predetermine sample size. Sample sizes were chosen based on previous literature and what is common practice in the field. No data were excluded from the analyses. The experiments were not randomized. Investigators were not blinded to group allocation of samples (genotype/treatment) during most data collection and analysis. However, key findings (chromatin reorganization in fasting and RNA Pol I-AID) were reproduced by at least an additional independent investigator who was blinded to group identity.

All experiments were reproduced with similar results. The images in Figs.